JPH01501755A - Construction of synthetic DNA and its use in the synthesis of large polypeptides - Google Patents

Abstract

Methods for the production of large polypeptides containing repeating sequences of amino acids utilizing biochemical techniques, specifically DNA sequences coding for the expression of the large polypeptides. Systems utilizing exogenous transcriptional and tranlational regions to control the production of the large polypeptides are also provided.

Description

[Detailed description of the invention] Construction of synthetic NA and its use in the synthesis of large polypeptides The United States Government, as a result of assistance provided by the Department of the Navy to research that led to this invention, , has certain rights in this invention.

Cross reference of related output i1 This application is filed under U.S. Patent Application No. 927,258, filed November 4, 1986. Part a of the issue! This is a continuation application.

The present invention relates to high molecular weight polymers, nucleic acids, or peptides that are expression products of the nucleic acids. in particular for use as a structural material. Production of high-molecular-weight tuptide containing a repetitive sequence by a biochemical method .

background Crude recombinant DNA technology allows for the isolation of native genes and their production in diverse host cells. It has been used for gene expression. This technique typically reduces the amount of energy that would otherwise be useful. biologically active substances such as interferon or peptide hormones that cannot be manufactured It has been used for the production of polypeptides. In addition, isolation of natural genes, and site Modified proteins can also be produced by specific in vitro mutations. It is possible to modify these genes and thus change the polypeptides produced. be able to. Created by combining various natural gene fragments There are also polypeptides, which are new polypeptides that are chimeric molecules of several naturally occurring molecules. Peptides have been created.

With the advent of efficient and automatic methods for chemical DNA synthesis, it is now possible to synthesize entire genes. It has now become possible to arbitrarily modify such synthetic genes during the synthesis process.

These various techniques are applied to the production of natural or modified forms of natural polypeptides. have used these techniques to create substantially new polypeptides. There have been very few attempts to do so.

By nature, polypeptides have a wide range of chemical, physical, and physiological characteristics. Ru. However, there are commercial uses for which naturally known polypeptides are not suitable.

Although biotechnology has become available, it is generally Applications have been limited to synthetic products and modifications of natural molecules. In contrast, organic The strength of chemical synthesis is that it can produce inexpensive carbon materials, including natural molecules, but most importantly is a polymer with defined and expected chemical properties that are unrelated to natural molecules. A wide range of species including completely new structures such as propylene and polyacrylates The point is that it can be converted into similar polymer molecules.

Such materials, especially high molecular weight polymers containing repeating sequences of amino acids, are manufacturing by means has proven difficult. repeating unit of amino acids The genes required for the production of large peptides are unstable and often undergo reactions within the gene. Intermolecular recombination occurs which causes deletion of the repeat unit. available through organic synthesis The development of biotechnology that creates polymeric molecules by biological methods is It will significantly expand the scope of biotechnology applications.

Brief description of related literature Cloning of up to 4 duplicate lactose operators in the same direction is 5adle R et al., Gene, (1980) 8: 279-300.

A hybrid introducer plasmid containing highly repetitive satellite DNA is called Brut. lag et al., Ce11. (Described in 1977>10:509-519 . The synthesis of poly(aspartyl-phenylalanine) in bacteria was reported by Doel et al. 　Nucleic　Ac1ds　Re5earch.

(1980) 8: 4575-4592. proline polymer How to increase proline content by cloning a plasmid encoding the production method The law is Kar+gas et al., Applied and Environment Described in Microbiology (1982) 43: 629-635 It was done. Highly repetitive that can be stably maintained within the plasmid replicon The biological limit on the length of the DNA sequence was established by Gupta et al. Described in o/Technology (pages 602-609, September 1983) It's been done Summary of the invention Polypeptides with repeating oligomeric units by expression of synthetic structural genes Provided are methods and compositions for the manufacture of. Coding the oligomeric peptide sequence The individual units coded are related to nucleotide sequences that take advantage of amino acid codon redundancy. and are diverse. A long nucleic acid sequence is a nucleic acid sequence that expresses a large number of individual repeat units. assembled by synthesis of oligomers, and those oligomers are combined A polynucleotide of desired length is provided. Significant expression of polypeptide product An expression system is used that allows the target host to grow to high concentrations prior to The polypeptide product is obtained in high yield, and it can be isolated from the host cells. obtain. In some embodiments, the transcription initiation system for the synthetic gene is activated by host RNA polymerase. is not recognized by the RNA polymerase and expresses a functional RNA polymerase under inducible control. A system is used in which the gene is contained in the host.

Brief description of the drawing Figure 1: Structure of niblasmid psY701.

Figure 2 = (A) β-lactamase or (B) gly-ala peptide Immunoplotting of polypeptides using antibodies.

Figure 3 is a flowchart for creating the Nibras Middle G10/Slp I.

Figure 4: Immunoplot of polypeptide products. (A) T by anti-Slp antibody 7gplO/Slp, (B) T7gp9/Sip with anti-Slp antibody, (C) Staining with Kumasi Blue.

FIG. 5 is a flowchart showing the creation of Niblasmid PSY856.

Figure 6: Time code showing the accumulation of kanamycin resistance gene product by the T7 line. vinegar.

FIG. 7 is a flowchart showing the creation of niblasmid pSY857.

FIG. 8 is a flowchart showing the creation of Niblasmid psY980.

Figure 9: (A) Containing the product of the β-galactosidase/S1pm gene fusion. (B) Immunoplasty of the same product with anti-Slp antibody. lot.

FIG. 10 is a flowchart showing the creation of Niblasmid pSY1285.

Description of preferred embodiments A novel polypeptide that is a polyoligomer of relatively short repeating amino acid units is provided. These oligomers are separated by spacers with different amino acid sequences. Sometimes they are connected. Therefore, this novel polypeptide consists of repeating amino acids are particularly useful as magnetic fiber proteins, including elastomeric proteins.

Genes encoding repeat unit-containing peptides are particularly genes containing duplicate repeat units. Configured to avoid j while previously associated with.

The gene of the present invention is a polymorphism of multiple DNA sequences encoding the same amino acid sequence unit. Consists of quantities. Here, two or more different polynucleotides encoding various amino acid units are used. The polymers may combine with each other to form a block copolymer. The individual units are , 4 to 30 amino acids (12 to 120 nucleotides), more commonly contains 4 to 25 amino acids (12 to 75 nucleotides), especially 4 to 25 amino acids (12 to 75 nucleotides) It has between 8 and 8 amino acids. And most of the time, the same amino acids are in the same unit. Appears at least twice, usually separated by one or more amino acids. Same amino Multimeric units encoding acid sequences contain redundant codons that achieve identical amino acid sequences. Depending on length, more than one type of nucleotide sequence can be included.

In most cases, the DNA composition of the present invention can be represented by the following general formula.

Kk (W) JXxNYy) i L 12 formulas, K can be any sequence of about 1 to 100 amino acids, generally 1 to 60 amino acids. Each DNA sequence that encodes the amino acid sequence of a amino acid, approximately 2 of the total number of amino acids. Less than 0%, generally less than 10% of the total number of amino acids, and any sequence in particular, the natural sequence, where the structural genes of the multimer are in reading frame with other DNs. It is fused with A. K will have the transcription initiation methionine codon.

k indicates 0 or 1, W has the following general formula.

[(A)n (B)pcoq During the ceremony, A usually has at least one amino acid that occurs at least twice in the sequence A DNA sequence that encodes the same sequence of amino acids each time it appears, Generally about 12 to 90 nucleotides (nt), or more, in the repeating unit. It generally contains about 15 to 75 nucleotides.

Usually at least two different A1s, generally less than 10 A1s, generally less than 6 There are several different A's that encode the same amino acid sequence, but with fewer They differ from each other in one nucleotide residue, and there are as many as 10 nucleotides. Although the residues may be different, generally they do not differ by more than 6 residues from other A sequences. stomach. At least two different codons for the same amino acid, e.g. glycine GGC and GGA are used for .

n is an integer of at least 2, generally at least about 8, and less than or equal to about 250; Generally an integer less than or equal to about 200, frequently less than or equal to about 125, and sometimes less than or equal to about 50. Ru.

B encodes an amino acid sequence other than the nine amino acid sequence units encoded by the A unit. I) A NA sequence different from A, which is a linking unit between oligomers of A units. functions as B generally has about 3 to 45 nucleotides (1 to 15 amino acids), more typically about 3 to 30 nucleotides (1 to 10 amino acids).

The B units appearing in the genes are the same or different, and generally there are more than 10 B units. from 1 to 45 B units, more generally from about 5 Different B's differ by 1 to 15 nucleotides and can be the same or different. can encode the amino acid sequence that has become.

p is 0 or 1 and can be different for each successive A unit. Ru.

q is an integer of at least 1, the number of nucleotides of A and B, and n and varies with the value of p. The variable q is the nucleus of the multimeric part of the structural gene. at least 90, preferably at least about 150, more preferably is at least 450, and most preferably at least 900. It will be selected. and the number of nucleotides generally does not exceed about 10,000 and more Generally no more than about 8,000, usually in the range of about 900 to 6,000, but generally It will be in the range of about 900 to 5,000. and, M is an I)NA nucleotide sequence of 0 to 18 nucleotides, Generally, A and/or left are limited to the amino acids of B, but usually A and/or Any amino acid sequence that includes the amino acids of B can be encoded.

X may be the same as or different from W, but in general they are different, and the following It has a general formula.

[(A')n' (B')p']q', in the formula, At, n', B', p' and qj are each ASn.

B, p and q may be the same or different, but at least one is different. , where similar symbols fall within the same definition as their corresponding symbols.

X represents 0 or 1.

N is defined the same as M, although it may be the same or different.

Y may be the same as W or may be a different salt, but in general they are different and have the following general formula: It is expressed as

[(A”)n”(B”)p’]q2 During the ceremony, A”, B2, B2, p and q2 are the same as As ns Bsp and q, respectively. They may be the same or different, but at least one is different and similar therein. Symbols fall within the same definition as their corresponding symbols.

y represents 0 or 1.

i is from 1 to 100, generally from 1 to 50, more generally when X and y are 0; represents 1 to 30, particularly 1.

When X or y is 1, the sum of q, q' and q2 is at least 2, generally It will be at least 5, and no more than about 50, and usually no more than about 30.

The total number of nucleotides is at least 90, generally at least about 150, preferably Or at least about 900. In addition, it may be 20 kg, Generally, about 12, and more typically less than about 10 are also possible.

The polypeptide encoded by the above DNA sequence has the following general formula: Probably.

K' k' (W' M' Xx' N"Yy'>ilJ" in the formula, Wo has the following general formula.

[CD)n(E)plq During the ceremony, D is the amino acid sequence encoded by A, and therefore one amino acid has numerical limitations based on the three nucleotides that define the codon that codes for Ru.

E is the amino acid sequence encoded by B and therefore defines a codon. Each E has a numerical limit based on the three nucleotides that define the code for B. They may be the same or different depending on the situation. And K”, W’, M’, X’ , N'', Y'', and Lo are WSM, X, N, respectively.

and the amino acid sequence encoded by Y. However, in K and L By subsequent processing such as protease treatment and cyanogen bromide treatment, Partial or complete removal of N- or C-terminal non-multimeric chains may occur. Ru.

n% ps qs ks is and l are defined in the same way as above.

A specific polymer composition having repeating units of multimers having the same composition (a) is as follows: It is represented by the general formula (X and y are 0).

Kk'[(D)n(E)pcoqLl'' in the formula, All symbol definitions are as above, and this DNA sequence has the following general definitions: has the formula

Kk [(A)n(B)pl qL1 During the ceremony, All symbol definitions are as above.

Characteristics encoding copolymer compositions having repeating units of 2 to 3 multimeric blocks A specific DNA sequence is represented by the following general formula.

Kk (W"M"X"N'Yy')i'Lf complete set, Wo is a multimer having the following general formula.

[(A’)n’(B’)p’]q’ During the ceremony, A3 is 4 to 8, generally 4 to 6 codons, and otherwise similar to A. Follow definition.

B3 is about 2 to 12, generally 2 to 10.

B3 represents 2 to 8, generally 4 to 6 codons.

p3 is 0 or 1.

q3 is about 2 to 25, generally 2 to 20.

Xo and Yl may be the same or different from W'' and follow the same definition as W''. cormorant.

Mo and No follow the definitions of N'' and Mo.

i” is at least 2, typically at least 5, and less than or equal to about 75, more typically It is about 50 or less, usually 30 or less.

Other symbols are as described above.

Generally, the compositions of the invention will have at least about 5 kDa, usually 10 kDa, preferably has a molecular weight of 15 kDa and 400 kDa or more can have a molecular weight, typically less than 300 kDa, more typically about 250 It is possible to have a molecular weight of kDa1 or less. Generally, the higher range is , a combination of multimers, each multimer typically having a molecular weight of less than about 150 kDal. droplets, usually less than about 100 kDa.

The nucleotide sequences used are, as mentioned above, because the repeating units are the same amino acids. will be synthesized with different codons. Usually code repeating units At least about 25%, more usually at least about 40%, of the nucleotide sequence Generally at least about 60% will be the same, but no more than about 95%, preferably In other words, it is desirable that about 90% or less be the same. Recombination where the initial construct is natural If it is shown experimentally that a phenomenon occurs, a large diversity within these ranges It will be used.

Of particular interest is the repeating unit 5GAGAG (G=nibrisin A=arani S = serine)

This repeating unit is found in naturally occurring silk fiber proteins, which include G AGAG (SGAGAG) - Represented by 5GAAGY (Y = tyrosine) . In the present invention, the repeating unit may have an N-terminus different from that of the repeating unit. λ4GAGAG, or generally at least about 3 amino acids, usually at least about 5 amino acids, more usually 12 amino acids, but 200 The following are designed to be other sequences, usually having 100 amino acids or less: . The N-terminus is usually different in a way that results in the expression of the fusion protein. This may be the result of inserting the gene into the vector by the method. desired characteristics of the product Any non-cancelling protein can provide the N-terminus described above. Special Endogenous host proteins, such as bacterial proteins, can also be used. Tan The choice of protein may depend on the nature of the transcription initiation region. Similarly, the C-terminus is , may have an amino acid sequence different from the repeat sequence. Conveniently, 1 to 10 There can be 0, usually 1 to 25 amino acids present, which are naturally occurring. can also be C-terminal with an existing structural gene, typically also a fusion product. arises from the formation of

The gene for silk-like protein (Slp) has approximately 5 to 25 repeat units, as described above. More usually, they can be prepared by providing oligomers of about 10 to 20 repeating units. Wear. Oligomers are linked by having different sticky ends, and two or more oligomeric units, generally no more than about 50 oligomeric units, more generally no more than about 30 of oligomeric units, and frequently less than about 25 oligomeric units. can be done.

Silk-like proteins have the same or different tendencies (hande+jness) This can be varied by having alternating multimers. For example, in the above formula (B) p can be an even or odd amino acid. In silk, glycine water The orientation of the methyl group and hydroxyl group of alanine and serine may be reversed. There is. (B) If p is an even number, use a continuous arrangement, if it is an odd number, (A) Alternate n. will take an array of . Therefore, (change the number of amino acids encoding Bin) Different properties can be obtained by doing so.

Of particular interest is the physical properties of carp (Bo+rlbyx mori) silk. and compositionally mimicking polypeptides.

G as the basic repeating unit that may be found in naturally occurring elastin VGVP (polypeptide having G=nibrisin ■=valine, P=nibroline Also interesting. In the present invention, the N-terminus can be any convenient sequence; If desired, it can be removed in whole or in part with a protease. general In addition, the N-terminal sequence that does not have the subject matter of interest may be less than about 100 amino acids, or more In general, it can be said that it is an amino acid with less than about 60 molecules.

Of particular interest are the basic repeat units of 4 to 8 amino acids and different internal repeats. about 6 to 20 amino acids, typically 8 to 16 amino acids about 6 to 10 units, preferably 8 basic positions, separated by a sequence of amino acids. It is a column. For example, the second repeat could be GAGAGS with two loose ends. cormorant. The total number of elementary repeating units is generally about 150 to 300, usually 175 to 300. It will be in the 250 range. The C-terminus may be a certain repeating unit or part thereof, or is another sequence of 1 to 100 amino acids, generally 1 to 30 amino acids. It can end with The C-terminus is not critical to the invention and is primarily for convenience. A wise choice will be made. Regarding the N-terminus, it is designed for proteolysis. Sometimes measurements are taken. As in the case of silk proteins, the elastin-like Similar operations may be performed on proteins.

Of particular interest is that it mimics the properties of elastin and imparts elastomeric properties. It is a protein.

Copolymers containing repeating units may have different units, the number of units in each multimer, their spacing, properties can be changed by appropriately selecting the number of repeats in the combinatorial assembly of multimers. It is a powerful means to make the world more popular. In this way, the number and arrangement of the blow mold mass bodies can be changed. A variety of different physical and chemical properties can be obtained by modifying the material.

An example of the use of block copolymers is the combination of set units and elastin units; A product that has different properties from a polymer that only has the same monomer units due to this. is obtained.

Various methods can be used to prepare structural genes. oligomer For the preparation, after hybridization, the duplex DNA with appropriate termini is Complementary DN Aa is synthesized so that A is obtained. If desired, it can be processed in a single step. Concatemers (concatemers) depending on hybridization conditions Each oligomer unit can be identical so that . usually , conventional annealing and ligation conditions, as described in the Examples below. is used.

If desired, the ends of the two units are complementary to the other unit, but the ends of the same unit are It is also possible to prepare two different oligomeric units such that their ends cannot be linked to each other. be. In this way, individual oligomeric units can be prepared and connected to each other. You can connect them to make a concatemer. In this case, the intervening concatenated sequences are Defined, at least in part, by the terminus. Depending on the configuration, the 5” end initiates transcription. A methionine codon may be provided, or the structural gene may contain a methionine codon at the 5° end. Adapters with unique sequences (possibly cleavable by specific enzymes) or a suitable linkage to provide a fusion product. can be inserted into a portion of the gene that is normally endogenous to the host.

A suitable complementary link between the adapter or truncated gene and the 5° end of the structural gene. By adding termini, these sequences can be ligated into the appropriate reading frame to create the desired Protein can be obtained. adapter or fusion protein The advantages of using it include binding, secretion, complex formation with other proteins, Two examples include QB-like sequences for specific purposes, such as affinity purification. It will be done.

Once a structural gene has been assembled, cloning becomes possible, especially for sequence lengths. A clone containing the desired gene can be isolated, and the gene can be extracted. and ligation to sequences for expression.

The expression construct contains transcription and translation components at the 5' and 3' ends of the structural gene, respectively. Translation start and end control areas will be included. As mentioned above, these areas can be created by using fusion proteins, in which the A structural gene is located downstream of and adjacent to another structural gene's start codon. will be inserted with the text and reading frame aligned. Alternatively, various transcriptional and translational initiation regions can be obtained from a variety of genes for use in the expression host, and as a result, the structural genes of the present invention These transcription and translation initiation regions can be modified according to the invention to obtain offspring transcription and translation. can be linked to the structural gene of A variety of termination regions are available, and these It may be from the same gene as the transcription initiation region or from a different gene. Many structures Similar to the adults described in the literature and used for other structural genes The methods described above can be applied to the genes of the present invention.

Of particular interest is the use of inducible transcription initiation regions.

In this method, the host strain is grown at high concentrations prior to significant expression of the desired product. obtain. Inducible transcription occurs, especially when the peptide is secreted by the host. It is more useful when retained in the host than when stored.

A number of inducible transcription initiation regions exist and can be used under certain circumstances . Its inducible region contains isopropylene for inducing the β-galactosidase gene. controlled by specific chemicals such as luthiogalactoside (IPTG) Can be done. Other inducible regions include the λ left promoter and the young , polycistro of various amino acids (e.g. histidine and tryptophan) genes, temperature-sensitive promoters, and regulatory genes (e.g., cl”857). Can be mentioned.

Another system that can be used to advantage involves the use of combinations of transcription initiation regions. legacy of hope control gene expression, but lack functionality for endogenous RNA polymerase. A first transcription initiation region, which is not functional in the expression host, is used. continue, The first transcription initiation region controls the expression of the RNA polymerase that acts on it. A second transcription initiation region, such as an inducible region, is used to induce transcription. In this method expression occurs after activation of control regions that regulate expression of exogenous RNA polymerase. It happens. Herein, we describe the transcription initiation region of T7 phage, in particular the transcription initiation region of T7 phage. This system is illustrated using the transcription initiation regions of genes 9 and 10.

Other systems grow based on changes in the environment such as lack of nutrients, temperature, osmotic pressure, or salt concentration. It depends on the usage of the mutant that undergoes the developmental changes. An example of this system is sporulation but not capable of producing components that initiate the expression of products involved in sporulation. Examples include grass strains. Therefore, by changing the medium conditions, the spore form The transcription initiation region associated with transcription will be activated. Under this situation, the host opens the expression The inducing agent, or activating agent, necessary for initiation is provided.

Various others that provide inducible control of transcription and translation of genes in specific hosts There is a method.

In most cases, the host is a prokaryotic or eukaryotic unicellular organism, e.g. bacteria, algae. , fungi, filamentous fungi, etc. Examples of hosts include Escherichia coli (E. coli), Grass fungus (B, 5ubtilis), B-stearothermophilus (B, 5te) arothera+ophius), S-cerevisi x-(S, cerev isiae).

・Expression 8 for expressing the target gene! Adults can act alone or with auxiliary agents involved in transcription. In combination with a gene, ligated into an appropriate vector for introduction into an expression host It would be common to do so. Along with other vectors described in the literature, numerous Vectors are commercially available. Typically, a vector has one or more unique restriction enzyme cleavage sites, replication threads for extrachromosomal retention in the host, and characterized by having one or more markers that allow selective pressure against the host.

Those markers are useful for complementation, prototrophy to auxotrophs, fungicides, e.g. It can confer resistance to antibiotics such as cancer or kanamycin. Rather than selective pressure, Marker genes are used that allow detection of specific colonies containing the gene of interest. In some cases, This situation is exemplified by the β-galactosidase gene. . In this case, a substrate is used that gives a colored product.

Expression constructs that include any auxiliary genes are particularly suitable for restriction enzyme cleavages, insertions, and and ligation into an expression vector according to known techniques.

The expression construct is then used to transform a suitable host.

If transformation or conjugation is used, whole cells or protoplasts may be used, depending on the host. Last can be used. DNA precipitated with calcium phosphate Nonionic detergents can be used to introduce plasmids into the host.

Because naked DNA can be introduced for integration into the genome, vectors can be used for host transformation. It is expected that there will be no need to use a tar. However, there are cases where embedding is desirable. However, it is preferable to use vectors as they provide more benefits. It is rare.

Depending on the nature of the vector, expression 4a! & the body is maintained on extrachromosomal factors, or integrated into the host. If integration is desired, prokaryotes and some For nuclear cells, it is preferable to have a sequence homologous to a sequence in the host chromosome. Yes. Generally, the sequence will be at least about 200 bp and about 5000 bp or more. The length is usually about 2000 bp or less. Although the selection of homologous sequences is somewhat capricious , complementation in which the host is an auxotrophic mutant and the homology confers prototrophy It can be used for

Transformants or integrants are grown at high concentrations in a suitable nutrient medium. After growth, transcription is induced according to the nature of the transcription system of the expression construct. Objective If the protein is retained in the cytoplasm, these cells can be harvested and lysed; Depending on the intended use, proteins can be processed by chromatography, solvent/solvent extraction, and after-treatment. Further purification can be carried out according to conventional methods such as identity chromatography.

Repetitive proteins find a variety of uses.

Slp protein plays a role in the production of fibers with unique aX, such as silk substitutes. can be used. Collagen protein can be produced and this collagen A gene may or may not contain a telopeptide depending on its function. Atero Peptide "collagen" is used as collagen for various prosthetics or other applications. It has immunogenic properties, making it a useful structural element for use as a substitute for Even if there is, it should be extremely low. Similarly, kerati with repetitive sequences Other proteins, such as proteins, can also be prepared according to the invention.

Other useful repeat proteins include spider silk and other repeat knot fiber arrangements. It can be prepared based on. Artificial peptides useful for immune sensitization are also used to treat parasites, Based on repetitive sequences present on various surface antigens of pathogens such as bacteria and viruses. It will be prepared as follows.

Example I A. Small-scale niblasmid DNA was prepared by the boiling method or alkaline lysis method [Maniat is et al., Molecular Cloning: A Laboratory Manual, Co1d Spring Harbor Laborator y, Co1d Spring Harbor (19g2>), 1. It was prepared from a culture of 5-.

B, Large-scale niblasmid-bearing strains were incubated with Luri II2 supplemented with appropriate antibiotics. a Growing overnight in broth. Cells were centrifuged at 10,000×G for 5 minutes. collected and diluted with 10-f ice-cold TE (10mM Tris-HCI pH 8, i cnM EDTA). Centrifuge the cells again and add 4@1 TES (TE and Re-suspended in 25% w/v Shorea:) and homogenized by vortexing.

Samples were stored on ice until the next step. Lysozyme (1- of 10 μ/-) was added to the cell suspension. incubate for 5 minutes before adding 2-0.5M EDTA (pH 8). Incubated. After 10 minutes of incubation, the 50-f protein Inase K (40 μ/−) was added. Subsequently, after 10 minutes, the 15-' Solution (0.1% Triton X-100, 1mM EDTA, 50mM T ris-HCI p) 18) was added. After 15-20 minutes, remove the cell lysate for 35 minutes. Centrifugation was performed at 000X G for 90 to 120 minutes. 20g of supernatant (19,8-f) Plasti containing CsC1 and ethidium bromide (10■/'), 400p1 Transferred to a stock test tube. After dissolution, the mixture was divided into two polyaromer ultracentrifuge tubes and heated. After sealing with It was 6 times. The lower plasmid DNA band was removed from the tube with a syringe needle. Bromide Ethidium was extracted three times with equal volumes of NaCl saturated isopropanol. 2 volumes of 8. 0 was added to the DNA solution and the DNA was subsequently precipitated with ethanol.

2.2 Preparation of main DNA Culture of JM 103, OD. . Proliferate until approximately 0.2, and Divided into recoats. Each aliquot was infected with fresh plaques of M13, approximately 6 Incubation was performed at 37° C. with vigorous stirring for an hour. Then, pellet the cells. The supernatants were preserved for subsequent infection. Two main phage DNA, Extracted by the boiling method (Janiatis et al.).

3. Protein removal treatment Perform phenol extraction on a convenient amount of DNA sample (usually 100eZ to 10d). went. DN A sample, O, OIM Tris-HCI (pH 7, 5) , an equal volume of phenol diluted with EDTA and saturated with H2O was added. This sample Vortex briefly and place on ice for 3 minutes. After centrifuging for 3 minutes in a small centrifuge, remove the aqueous layer. into a new test tube and re-mix equal amounts of chloroform:isoamyl alcohol (24 : Extracted with 1).

4. Ethanol precipitation DNA in dilute warm solution was concentrated by ethanol precipitation.

The DNA sample was treated with 1710 volumes of 3M sodium acetate (pH 7,5) and 2-3 volumes of Cold ethanol was added. Incubate this DNA at -70°C for 30 minutes or at -20°C. Sediment overnight and pellet by centrifugation for 15 minutes at 4°C in a small centrifuge. did. Wash the pellet once with 200 m cold 80% ethanol and again at 4°C for 10 min. pelleted for a while. After air-drying or freeze-drying, resuspend the pellets in an appropriate buffer. Ta.

5. Phosphatase treatment of DNA Phosphatase treatment of DNA involves adding 14 (25 U) of U to the restriction enzyme digestion reaction product. Direct addition of intestinal phosphatase (Boehringer! annheim) This was carried out by continuing the incubation for 30 minutes at 37°C. child The phosphatase of Inactivation was performed at ℃ for 60 minutes.

6. Fill-in reaction DNA with DNA polymerase I, 50m! JTr is-HCI (p) 17, 4), 50d KCI, 5 (C!J MgCh, and resuspended in a buffer containing 400 volumes of each of the four deoxynucleotide trisulfates. It got cloudy. Add 10 units of Klenow DNA polymerase (BRL) and incubate for 15 minutes. The reaction was allowed to proceed at room temperature. Next, this DNA was extracted with phenol and extracted with ethanol. precipitated.

7. The T44 polynucleotide kinase reaction includes the following: . T4 polynucleotide kinase (BRL), 150nH DNA, 1p! 1 0x Kinase warm solution [0,7! Tris-HCI (pH 7,6), 0.1 MgC1,,50mlJ DTTI, and [Co’P]ATP (200 ~300 nCi). This was incubated at 37°C for 30 minutes to remove the DNA. was purified using a NACS column (Bethesda Re5each Labs). Manufactured.

8. Digested DNA with restriction enzyme endonuclease is diluted with lx'AA'' buffer solution [1 0XAA buffer = 330CIM Tris-acetic acid (p) 17.9), 660d vinegar Potassium acetate, 10hM magnesium acetate, 50mM dithiothreitol (DT T) and 1/d bovine serum albumin (nuclease free). Digested with enzyme endonuclease (BRL). Reduce the concentration of DNA as much as possible IR/kept below 25d. Incubation is Bal I, Ban I , and restriction endonucleases except NaeI (these digestions were done overnight) Most of the tests were carried out at 37°C for 1 to 4 hours.

9. Agarose gel electrophoresis for DNA analysis. Add 0.2 to the DNA sample for gel analysis. volume of loading warm solution (5x electrophoresis warm buffer, 0.01% bromophenol blue dye) , 50°C MEDTA, and 50% pricerol) were added. Then 1.0 % (W/V) agarose gel in a horizontal submerged electrophoresis device lane. . Electrophoresis buffer was either IXTAC or 1/2XTBE. I XTAC consists of 40n% MTris-base, 10mM EDTA, diluted with acetic acid. pi('?, 81:: adjusted. 1/2XTBE is 0.045M Tris- Base, 0.045M boric acid, 1 tr, M EPET (pH 8>). gel After being energized at 40 to 50 V for 18 hours, it was removed from the device and 0.5 dl of bromide Stained with tidium for 30 minutes. DNA bands can be visualized by long-wavelength UV irradiation. It became.

10. Preparative agarose gel electrophoresis Methods and materials are similar to those for analytical agarose gel electrophoresis. the only difference Use a low melting point agarose gel and adjust the concentration depending on the size of the DNA to be purified. The point was to use it in the range of 0.5 to 2.5%. DNA restriction enzyme fragments are After visualization with tidium, it was excised from the LMP agarose gel.

11. NAC3 purification The gel fragment containing the DNA was melted at 70°C for 5 minutes and placed (10 ml of Tris- It was diluted approximately 5 times with HCI (pH 7.5), 0.2M NaC]. this gel The solution was applied to a NACS column (BRL). Wash the column with the same buffer of 5- Ta. The combined DNA is T E 2 for a 1000 bp DNA fragment. C10mM Tris-HCI (pH7,5), 1.0NaC1) 300 d, for larger fragments TE 3 [10mM Tris-)ICI ( pH 7.5), 2M NaCl] eluted at 300 m. The eluted DNA was Concentrated by ethanol precipitation.

12- DNA ligation reaction The following was used for the ligation reaction of DNA sticky ends. 1 tail DNA, 1xAA buffer solution (see 8. above), 1 mM ATP, and 20 units of T40NA Rigger (BRL) to a final volume of 20I. The DNA ligation reaction was carried out at 15°C with 16-1 It was allowed to proceed for either 8 hours or 1-2 hours at room temperature. DNA with blunt ends For the ligation reaction, lar DNA, 25+++M Tris-HCI (pH 7, 5), 5mM MgC1z, 5mM DTT, 0.25mM spermidine , 200 mg BSA, 1111M cobalt chloride hexamine (HCC), 0 ．． 2 containing 5mM ATP and 400 units of 74 DNA ligase (NEB) A reaction solution of 0111 was used. DNA ligation reaction is performed at room temperature for 30 minutes to 1 hour. It progressed for a while.

Bacterial transformation method Enterobacterium was inoculated. This is OD. . Culture with shaking at 37℃ until 0.5. Ta. The culture was placed on ice for 10 minutes and centrifuged at 6,000×G for 10 minutes. . The cell pellet was resuspended in ice-cold 0.1M MgCb 100 solution and placed on ice. The mixture was left standing on top for 30 to 40 minutes, and then centrifuged again. Pellet 2-100mM Ca Resuspend in C1, transfer to a sterile tube, and incubate on ice for 24 hours. Ta. Thereafter, the competent cells were aliquoted and stored and frozen at -70°C. An aliquot of the sample was thawed on ice.

Add 0.1 to IK DNA to 501!1 cells and leave the mixture on ice for 30 minutes. Incubated for a period of time. Take this test tube out of the water tank and place it in a water bath at 42℃. It was left standing for 2 minutes. Add L liquid medium (1d) and keep the transformation mixture at an appropriate temperature (usually , 30°C or 37°C) for 2 hours. 1710 of this transformation mixture was Place in a medium plate containing appropriate antibiotics, and add XGAL and IP as necessary. TG was added.

3. DNA transformation of Bacillus subtilis Bacillus subtilis cells are grown to an initial steady state B (less than 5% change in klett units in 15 minutes). Made it grow. Transformation is determined (Anagnostopou et al., 198 1) According to (Reference 8). Cells (0,45-f) were added to 1-10 g of DN A was cultured with shaking at 37°C for 80 minutes. After that, T with appropriate antibiotics Plated on BAB agar plates.

4. Isolation of plasmid DNA from Bacillus subtilis Isolation of plasmid DNA from Bacillus subtilis , pelleted cells were added to solution 1 [: 50 d glucose, 10 mM EDTA] , 25ffIM Tris-HCI (pH 8), 10mg/af Lysochy alkaline lysis except that resuspended in Obtained by a method similar to the method. Subsequently, solution 2 of 16- (0,2N NaOH, 1% (w/v) SDS) was added and incubated on ice for 10 minutes. most Afterwards, 12-3M potassium acetate (pH 4,8) was added and incubated on ice for an additional 20 minutes. Incubated with Lysed cells were placed in 5 orval SS rows for 15 minutes. The mixture was centrifuged at 15.00 rpm in a rotor. DNA was prepared with an equal amount of isopropanol. The mixture was precipitated by addition and further centrifuged at 7.00 rpm. 5 pellets 10d Tris-HCI (pH 7,5>, 1mM EDTA (TE ). This solution was subjected to one phenol extraction and one chloroform extraction. We carried out the DNA was ethanol precipitated and resuspended in 3 dl TE. 4 ．． Addition of 2 g of CsC1°10/- ethidium bromide 400d and TE The capacity was adjusted to 5.2 d. This solution was added to Beckmanquicks. Transfer to a polyallomer centrifuge tube and use a Beckman vti650 Centrifugation was carried out at 45.00 rpm for 18 hours in a microtor.

sequence (GAGAGS), synthetic peptide of GAAGY, Kagen and G5 Coupled to BSA using the glutaraldehyde method of 1ck (1979). Ta. The extent of coupling is tracked by trace amounts of radioiodinated synthetic peptides. did. O using the peptide conjugate at a concentration of 1/d in complete Freund's adjuvant. Rabbits were immunized with Bk. For rabbits, use 30-day Freund's incomplete adjuva. The antigen dissolved in the patient was reinjected, and the titer was measured on the 60th day. Synthetic peptides as antigens Positive serum was detected by microtiter RIA using Peptide. rlAe thods of Radioiciunoassy J, Kagen and G11ck (1979), Jaffe and Berman (i4), Academic Press, p328.

51pI! A peptide consisting of 53 amino acids corresponding to the I sequence was The iED was prepared using a Biosystems peptide synthesizer. molecule ff1364 The yield of this material with 0 was approximately 0.5 g. This peptide was added to serum Coupled to rubumin. This substance is used for antibody preparation in rabbits. , Antit++dies, Inc.

was sent to. Antiserum was obtained.1. This is a synthetic pair of 51pI and Slpm sequences. Reacted with putide. These antisera contain a fusion peptide containing the gly ala sequence. It is useful for detecting

According to the method described above, the general formula (V-P-G-V-G). An antigen with Ta. This was coupled to the hemocyanin of keyhole linbet. Continued A polyclonal antibody conjugated to the ELF peptide was prepared as described above.

2. Polyacrylamide electrophoresis of proteins obtained from growing cultures of approximately 10 ' E. coli was pelleted by centrifugation at 10.0 OOx G for 5 minutes. cell pellet sample buffer [100d Tris-)I at 100 to 500J=f C1(p)16.8), 4% SDS, 10% β-mercaptoethanol, 6 0% glycerol or sucrose] using a Tekr+er sonicator. and ultrasonicated for 30 minutes. Boil the sample for about 5 minutes, then add 20 to 100 I Cell lysates were subjected to SDS polyacrylamide electrophoresis (7.5 to 16% w/v). I put it on. This gel was prepared by Laemmli (Nature, 227:680-6 85 (1970)).

The proteins in the gel were purified with 2% Kumasi Blue in 10% methanol, 7.5% acetic acid. Stained for 1 hour and destained overnight in 10% methanol, 7.5% acetic acid.

3. Immunoplot of proteins in gel After protein electrophoresis, remove the gel. One of the sandwiched glass plates was removed from the polyacrylamide gel. gel surface Transfer buffer (25mM Tris-HCI, 192.+M glycine , 20% methanol). Nitrocellulose paper strips (Sartorius , S! 11307) was saturated with transfer buffer and placed on the gel. centre Air bubbles between the filter and gel were removed. Gel and nitrocellulose filter J Zogen Toku Bh (D) Lance 7 arm unit (Bio Rad) Ta. Transfer was performed at 200 mA for 3-4 hours. Next, a nitrocellulose filter - Take out Amido-Schwartz (0.05% Amido Black, 45% deionized water, 45% methanol, 10%! ￥ acid) for 3 minutes. Fi “BLOTTO” (5% w/v nonfat dry milk, 50 mlJ Tris-HCI pH 7,4,0,9%w/vNac1.0. 2% sodium azide). This filter is 0. 5 x BLOTTO (2,5% w/v skim dry milk, 50mM Tris- HCI pH7,4,0,9% w/v NaCl1,0,2% sodium azide Place it in serum diluted appropriately (1:50 to 1:500) with The mixture was slowly stirred for 6 hours. Filter with TSA (50r+; M Tris- HCi pH7,4,0,9%w/vNac1.0.2% sodium azide) Washing was performed for 1 hour, changing the area 5 times. Set this boot to IX 10’cpm 0.5 x BLOTTO liquid 15 containing +2s knee protein A - medium liquid 5, The mixture was slowly stirred at room temperature for 2 hours. While changing the filter at least 7 times, Washed for 2 hours, rinsed once with deionized water, and air dried. This plot is based on Saran Cover with plastic wrap and autoradiography/U composition Henrikson Determined by the PTC derivative Ljl''A method of Meredith (1984) Ta. Protein samples were incubated in vacuo for 24 hours using 5.7N constant boiling HC, f. Hydrolyzed at 108°C. After reaction with PITC, the amino acid derivative is Lett Packard 1090 system and 5upelc + C18 color Reversed phase high performance liquid chromatography with was detected at 254 nz. At this time, the mobile phase is 0.1!　NH,O Elution was performed using a linear gradient of Ac+50% acetonitrile. Henrik son.

RoL, and Meredith, S, C, (1984) Reverse-phase high-speed liquid cooler. Amino acid analysis by chromatography J Anal, Biochem, 137 :65-74゜5, amino acid sequence analysis The N-terminal amino acid sequence is from Applied Biosystem Model. by automated Edman degradation using a 470A gas phase protein sequencer. It was decided that PTH amino acid derivative is Hew 1ettpackard 10 Reverse using 90 system and Altex C18 column (21JX25) It was analyzed by phase high performance liquid chromatography. For elution at this time, a complex gradient is used. A distribution buffer system was used.

6. Peptide synthesis Synthetic peptides were prepared using standard symmetrical anhydride compounds provided by the manufacturer. Biosystem 1Jodel 430A peptide synthesizer solid phase Prepared by synthesis. The coupling yield of each step was calculated by Arin et al. (1981 ) was measured by the ninhydrin quantitative method. Synthetic peptides using anhydrous HF was released from the solid support and the amino acid protecting groups were removed (Stewart and Y. cUng.

1984). Crude peptides were chromatographed on Sephadex G50. It was desalted by 5arin, V, ni, Kent, S, B,) 1. .

Tan, J.P., 2 and Marrifield, RoB, (198i> , Anal, Biochem.

237:927936 o Stewart, J], and Your+g, J, D, (i984).

“Solid Phase Peptide Synthesis Method J, Pierce Chemical Compan y.

Rckford, IL, pp 85-98°N, N-diisobrobifuos Foamidide, adjustment hole glass force 51. , and all synthetic reagents were provided by Appli ed　Biosystems.

Obtained from Foster C1ty, California.

Synthetic oligonucleotides were prepared with a 10-fold excess of protective phosphoramide and synthetic support. Applied Bio system+s Mode) 380A DNA synthesizer Prepared by the ster phosphorous acid method. The method used for synthesis, together with the synthesizer It is preferred to use standard methods, which are known methods (Matteucci et al. and others.

Journal Amer, Chem, Soc, 103:3185-221 9 (1981)) o Deprotection and release of the oligomer from the support is performed by McB r ido et al.

Tetrahedron Letters, 24:245-248 (198 It was carried out according to the standard method described in 3). Repetitive yields of the synthesis are shown in Applied B For optical density measurement of eliminated protecting groups, recommended by iosystem;s (19g4) According to the report, it was over 97.5%.

The crude oligonucleotide mixture was published in Applied Bi, September 9, 1984. osyste:s protocol (U, ser Bulletin Nc13) Purified by preparative gel electrophoresis as described. acrylamide gel The concentration was varied from 10 to 20% depending on the length of the oligomer. Made by tfit Oligomers were identified by UV irradiation, excised from the gel, and subjected to the infiltration method (Smi Th-! eti+ocfsir+Enzymology, 65:371-3 79 (1980)).

2. DNA sequencing method The DNA sequence was determined by the following method. The fragment containing the region of interest was mp18 or M13mp19OJaniatis et al., 1982. and No. rrander et al., 1983). 1 piece Prepare ff1JDNA and use 35S-deoxyadenosine-5 as a label. ′ (α-thio)-triphosphate (New England Nocl ear) using the primer extension method (Sanger et al., 1977 and Bi ggin et al., 1983). Reverse transcriptase (ioiecL+ lar Genetics) was sometimes used for primer extension. this# Za:B; ursky et al. (Gene Anal, Techn, (1985) 2:89-94) dideoxy:deoxynucleoside triphosph The rate ratio was adopted. Deoxyadenosine tri labeled with 32p or ss3 Phosphate was used in these reactions. It is seen in sequences with a somewhat large amount of GC. The compression artifact is caused by deoxyguanosine triphosphate from the G reaction. was removed and replaced with deoxyinosine triphosphate (PL Biocb). emouth ica Is) at a final concentration of 37.5I! The problem was solved by using M.

In other formulations, the guideeoxy GTP concentration in the G reaction was set to 0.51.

All sequences were run on 6 or 8% polyacrylamide gels containing 8M urea. (Sar+ger et al., 197 g). The primers used for sequencing were , P　L'　Purchased from Biochemicals. For thetano storage and analysis , DNA5tar and International Bictechnology Software from both companies, Inc., was used.

3. Plasma containing sequences that are subject to in vitro mutagenesis of cloned DNA Sumid DNA (Ig) was digested in two separate reactions. In the first reaction , one or two restriction endonucleases that cut at sites adjacent to the region of interest was used. In the second reaction, one site is isolated from the sequence to be mutated. Digested by a restriction endonuclease that cuts only occurs in the first reaction The DNA fragments are separated by agarose gel electrophoresis and a large The fragment was excised and purified. DNA obtained from the second reaction, from the first reaction The resulting large NA fragment and the 20 to 30 bases in length contained in the mutant sequence of synthetic oligodeoxynucleotides were mixed in a molar ratio of 1:1:250.

The mixture contains 25 to 100i11 of 100mM NaC! , 6.5mM by heating at 100°C for 3 min in Tris-HCI (pH 7,5) Denatured. The denatured mixture is re-annealed by gradually lowering the temperature as follows: I let it go. 37°C for 30 minutes, 4°C for 30 minutes, and 0°C for 10 minutes. For the reaction, 0 ．． 5 d-deoxyribonucleotide triphosphate, 1111? J ATP , 400 units of 74 DNA ligase, and 5 units of colon WDN A polymer. lase large fragment was added and synkinivated at 15° C. for 12 to 16 hours. Continued The reaction mixture was transformed into E. coli and antibiotic-resistant colonies were selected.

Fermentation conditions The fermentation container is 151 Chemap (actual capacity ill　Ot2)　te;jof , :,.

The culture conditions were: temperature = 30°C, p)! =6.8, pH adjusted using NaOH2.5M I performed a section. The pressure at the top was less than 0.1 bar. The dissolved 8 elements were adjusted to 50%. It was knotted. The air flow was varied from 0.51/i+in to 201 t/sin. stirring The stirring speed was varied from 200 to 1500 rpm.

The fermentation vessel contained 10% (V /V) was inoculated.

Medium B was a fermentation medium. The starting capacity is 5I! Met.

When the glucose concentration reaches 1%, in order to keep the glucose concentration at about 1%, A concentrated solution of medium B (5X) was added.

The culture is OD. . = 60.0, the temperature was increased to 42℃ in 10 minutes. The temperature was then lowered to 39°C over 2.5 hours.

The cells at this time were harvested by centrifugation and frozen at -70°C until processing.

NaC110 Tryptophan 10 Yeast extract 5 Kanamycin 5X10-” NH4Cl 4.5 KH2PO,0,76 Mg5Oa ・7Ht0 0. i8 , So, 0.09 CaC1z 24X 1O-3 FeSO, 7H, 07, 6X 10-'T E O, 592 Casein amino acid 25 Yeast extract 5 Glucose 20 Kanamycin 5X10-” Example 2 An 18 bp oligopeptide that encodes the most frequently repeated oligopeptide in protein proteins. DNA was synthesized in vitro [1ucas, F- and M, Ru dall (1986) "Extracellular fiber protein": "Silk".

pp. 475-558, Cozpreher+5ive 8iochemistr y, Volume 26, 389M.

Florkin and F.) I, 5totz (eds.) Elsevier, Am 5terdai+], two single fl DNA were synthesized, and then the resulting Multimerize double-stranded segments head-to-tail, up to 13 repeats and beyond 13 repeats It gave rise to a funkatemer. Structural gene of 5ilkl that we are researching is the oligopeptide gag, gs (g = niblicin a = alanine, S = serine ), and this structural gene is called a "monomer". So Then, 2゛6 repeats (2 for Sip), 39 repeats (S1pI-3), 52 repeats ( SipI 4), 65 repeats (S1pI-5), and 78 repeats (SlpI-6 ) to create 31pI of “dimer, trimer, tetramer, pentamer, and hexamer” including Manufactured. There are short intervening sequences between each monomeric unit. The assemblage is as follows You can draw it like this.

Repeat D N A triple row 5'-GGTGCGGGCGCAGGAAGTCGCCC GCGTCCTTCACCA-5' One of the two shown above 23D N A front It was synthesized as described below. These strands were separated by gel electrophoresis. released, phosphorylated using T4 polynucleotide kinase, and mixed together to animate -Ring. As a result, two strands naturally oriented head-to-tail in a long concalamer. A chain segment resulted. The inter-segment phosphocyester bond is T4DN A ligase; thus formed. This reaction was performed using DNA polymerase Klenow. The fragments were used to finish by filling in the sticky ends. with blunt ends The repetitive DNA was transferred to the plasmid vector pUC12 [Veiera et al., Ger+ E 19:259-268 (1982)) at the old ncnRENB position.

The ligated DNA was transformed into one strain of E. coli HB i01, and this transformant was incubated. Selected by ability to grow in the presence of ampicillin. of a promising clone Size and orientation of DNA was resolved by REN digestion and gel electrophoresis. analyzed. The DNA sequence of the isolate with the large insert in the correct orientation was determined.

The “monomeric” clones selected for subsequent multimerization are oligopeptides′ It has 13 repeats encoding agagsg and was named psY708.

S] DNA sequence of pI insertion site, deduced amino acid sequence of SipI and RE The N site and the flanking regions of psY708 are shown in Table 2.

3. Construction of expression vector psY701 Plasmid pSP 65 (10x, Boehrtnger) Jannhe ia) was digested with Aatm, extracted with enol and precipitated with ethanol. D The NA was resuspended in 101 H2O. 172 of this DN A in the following mixture Digested with exonuclease.

DNA5■, IOX exonuclease■ buffer (60 0mM Tris-ICI pH 8, 6-6mM MgCl2.10mM β-Metal lucatoethanol), and 9 units of exonuclease■. 20iIl sample 01.1.2.5.5 and 7.5 minutes later, immediately injected with 5g of tRNA and A solution containing 36 units of S1 nuclease (30mM sodium acetate pH 4.5) ,0.25M with acl, lmMznS04) 100〆. Ink cartridge tion was carried out for 45 minutes at 30°C and added with 15jd of stop buffer [0.5M Tri s (pH 9,0), 125mM EDTA, 1% w/v SDS, 200 g/tj tRNA) to stop the reaction. This sample was extracted with phenol. , precipitated with ethanol. The resuspended DNA was added to Sal! Digested with lI REN and electrophoresed in a 1% low melting point agarose gel. β-lactamase gene , the plasmid origin, and the DNA carrying the β-galactosidase promoter Fragments were excised from the gel and dissolved at 65°C. Added 1 volume of H2O. of each sample The DNA was recircularized by DNA ligation in the presence of agarose. this reaction For this, melt gel 8iL1, DN A ligation reaction buffer [100a+M Tri s-HCI (pH 7,5), 50d MgC1z, 50mMDTT] 2 Contains 10 units of T4 DNA ligase and 3 hours incubation at 15°C. I went to JM 101 competent cells were transformed with the ligated DNA and transformed. Transformants were grown by growth on medium plates containing ampicillin (40χ/-Z). That's what I chose. Plasmid DNA was prepared from four transformants. This DN Digest A with BaaiHI and use the Klenow fragment of DNA polymerase 1. Label with P-dGTP, digest with PvuI, and then gel purify the smallest fragment. Ta. Fragments from one transformant were arranged by the Maxim-Gilbert method. The row has been decided.

The other three plasmid fragments were further digested with Taq I and electromagnetized on the same gel. It was pneumophoresed. Sequenced plasmids contain overlapping cloning sites and β-lac It had a fusion between the N-terminus of tamase and a position upstream from flATG. Other The size of the BamHI-TaqI fragments of the two plasmids is such that the fusion Duplicate cloning site of lactamase gene and 4th r order of β-lactamase gene It was shown that this occurs between the amino acids of N of altered β-lactamase The DNA of the terminal region and the corresponding amino acid sequence were extracted from the REN site of psY70i. It is shown below along with a circular map (see Figure 1). The amino acid sequence in Figure 1 is as follows: .

met-t h r-me t-i 1e-t h r-pr o-5er- 1eu-g 1y-cys-a r g-ser-th Chichi@- 1eu-g 1 u-asp-pro-h is-phe-arg-va 1- a 1 a-1eu-i 1e-pro-phe-phe-ala-a) a-p he-cys-1eu-pro-val-phe-ala-his.

4. Insertion of monomer J 51pI from psY708 into psY701 plasmid 70 g of psY was digested with old ndI[I, and the Klenow fragment of DNA polymerase I and digested with BaffII. plus MidopsY701 was digested with XbaI, filled in as above, Then, it was digested with Baff1HI.

Next, the DNA fragment from psY70g and the main sequence of psY701 were transferred to a low melting point agar. Purified by gel electrophoresis and then purified by NAC3 (BRL) column. Ta. Appropriate fragments were mixed, ligated, and transformed into E. coli JM 109. . The transformed cells were treated with ampicillin (40 mg/m), IPTG (5x Contains 10-'M)b and That's what I chose.゛Analyze the plasmid content of the transformants for further investigation. One (pSY756) was selected for this purpose. This is due to the mapping of the REN site. If determined by This is because the. The entire DNA sequence of pSY756 has not been determined, but the insert The connection site between the vector and the upstream Xba I, and the downstream Bam) I The restriction enzyme cleavage sequence was proven to be accurate.

5. REN the multimerization plasmid psY708 of the 51pI gene of pSY756. Digested with Sc+a I to obtain polypeptide arz (a la-g 1y-a 1 a-g ly-set-g ly) + athr-1eu-g ] ] u- The code sequence for asp-pr(R<AGAGSG), , TLEDP) is The supported DNA fragments were purified according to 4 above. Plasmid pSY756 5 11a, deproteinized, and then purified from psY708. It was ligated with a DNA fragment. E. coli J! The transformant of J109 was treated with ampicillin. Selection was made on medium containing. The various clones have the same monotony as the psY708 clone. 2 units (dimer pSY882), 3 units (trimer pSY883), and and 4 units (tetramer pSY884). Similarly, pentamers and A hexamer was also constructed. All of these peptides are genetically stable and β- β-lactamase produces peptide g1y-ala as a fusion with lactamase Expression of the S]pI peptide in E. coli cells as a fusion protein with Detected by Noplot (Western) analysis. Anti-rS1pJ antibody , produced for synthetic box proteins. As shown in Figure 2, this The antibody is a 5lpl dimer and a 3M form fused to E. coli β-lactamase. I reacted. Insertion 8 of 5lpl follows the fifth amino acid of the signal sequence of this enzyme. . β-lactamase antibody (Figure 2A) Similar to proteins, the protein that appears at approximately 28 kD as the main antigen band in this diagram Processed mature enzyme was also detected.

The mobility of all Sip I, including polypeptides, is extremely small, and this The proteins are not large enough to be seen on the gel.

Anti-Sap antibodies are also effective in detecting these fusion products.

Lanes 2 to 5 of Figure 2B are differential proteins containing a dimeric fusion of β-lactamase and 51pI. Four clones are shown. whereas lanes 6 and 7 contain the trimeric fusion It is from two clones. Apparently, the antigenicity of the trimer is greater than that of the dimer. Remarkably strong. From previous experiments, the fusion protein containing only the monomer of 51 pI was It can be seen that it was not detected at all by the anti-Sip antibody. Antigenicity of trimeric peptides Enhancement detects fusion with β-lactamase signal bebutide . The processed form has a position of approximately 33 kD in lanes 6 and 7 of Figure 2B. It can be seen in the location. Normally processed β-lactamase mature enzyme (β-lactamase) (reacted with enzyme antibody), and the signal of trimeric Slp I-3 and β-lactamase. The peptide appearance corresponding to the fusion between the 5lpl peptides is the 5lpl arrangement within the signal sequence. Despite column insertion, normal proteolytic processing of this enzyme occurs in the large intestine. This suggests that it is caused by bacteria.

7.a, Expression of 5lpl gene by fusion to T7 gene The 51pI sequence is In bacteria, a fusion protein with gene 9 and gene 10 of batatteriophage T7 It also appeared as a quality. This configuration is schematically shown in FIG. Plasmid psY9 15 (containing the tetramer 51pI-4) was completely digested with REN Sal I. and partially digested with Ba+n)I. D containing tetramer Sip I-4 The NA fragment was purified and digested with RENSal I and Bam) I Plasmid psY114 (Promega Biotech pG2) Clone inside. From this intermediate plasmid, a 51 pI tetrameric insert was It was excised with ENAcc I and EcoRI. Furthermore, this fragment is pSY633 digested with coRI and AsuII [: complete T7 gene pBR322 containing the sequence: pAR441 of 5tudier et al. (1986) ) was cloned into. In the obtained plasmid, 5 lpl of the tetramer is the gene It is fused to the translation reading frame of gene 9 near the 90C terminus. IPTG inducible T7RN inserted into the chromosome under the transcriptional control of the β-galactosidase promoter E. coli strain 0-48C5 containing the A polymerase gene tadier et al., 19 This plasmid was used to transform 86 strain HMS174 (λDE3)]. was used. In this gene arrangement, expression of the SlpI-4 sequence is T7R itself regulated by the G-inducible β-galactosidase promoter Depends on the production of NA polymerase. As shown in Figures 4A and B, When these cells are induced with IPTG, the gene 9/Sip I-4 fusion gene The protein product of the gene is synthesized and synthesized by the antibody to the Sip Milpeptide. Detected. The fusion product migrated through the gel as if it had a size of 82 kD. move. The expected size was 65kD. This peculiar transfer mF2 is an example of A characteristic of the external amino acid composition (rich in glycine and alanine) that found in Slp-containing products.

Similarly, the protein promoter region of T7 gene 10 and the T7 gene Plasmid pSY63g containing the first 13 amino acids of the 1o protein (Sta dier pAR2113) was digested with RENBam) II, and D Fill in with polymerase Klenow fragment and digest with REN EcoRI did. This linearized plasmid contains the tetramer 51pI-4 of pSY633. The AsuII-EcoRI fragment having the following was cloned. This ligation reaction As a result, an in-frame fusion of the silk tetramer following the 13th amino acid of T7 gene 10 occurs.

The latter fusion product is used for spindles without any processing. They are N The terminal 13 amino acids are only a small part of the large Sip I protein. It is from. The fusion product is approximately 30 kD in size but has exceptional mobility. Then, it moves as if it were 50kD. This is shown in FIG. 4A. plus mid pG9/Sip I-4 and pG10/Slp I-4 express T7 Insert the kanamycin metabolic gene into the β-lactamase gene in the opposite direction to the system. It was further improved by this. Therefore, any low level Expression also does not lead to an increase in β-lactamase activity. With such activity, Ampicillin added in the medium was removed to screen for lasmid retention. . When ampicillin was deficient, the plasmid disappeared from the culture. Kanamai Syn resistance genes circumvent this problem and are useful in T7 expression systems, especially in large-scale cultures. This is a significant improvement. The kanamycin resistance gene (originating from Tn903) is H Plasmid pUc4K (Veira, J. & LMe ssir+g, 19g2. Gene, 19:g59-268) isolated p E. coli strain 0-48 carrying sY997 was incubated with Chezap or Braun's fermentation. Using a container, the OD (Klett unit) is 300 (3X10 "cells/-f)" was grown at 37°C.Furthermore, 3.5 d of IPT G was added to induce the T7 line.

After 150 minutes, transfer the cells to a Millipore filter unit (Pellicon cassette). System, filter exclusion limit molecular weight 100,000) to concentrate 10 times. did. Subsequently, the cell suspension was frozen at -70°C until processing.

The cell suspension was lysed in a 42°C water bath and lysed in a French press.

The lysate was centrifuged at 125.000xG for 1 hour at 25°C. D the transparent upper groove Treated with NAase (250-w/td) for 15 minutes at room temperature. continue, Filtered through a 0.457- sterile filter. Measure the volume of the filtrate and stir warmly. while incubating on ice. In addition, 231■ ammonium sulfate was added to each 1t7 portion of the filtrate over a period of 45 minutes. For each 10g of ammonium sulfate laf of Na0i1 was added to neutralize the po. After 2 hours of continuous stirring, mix The object was spun at 9.000×g for 10 minutes. Return the pellet to the original filter using distilled water. It was resuspended in 1/10 of the liquid. Centrifugation and resuspension were repeated three times. pellets, The raw filtrate was resuspended in distilled water. For each sample, protein concentration, Amino acid composition and amino acid sequence were determined using standard methods. This gives the product This is one of several ways to do so. According to this method, the purity of 51 pI-4 was obtained. As expected, the amino acid composition is almost entirely comprised of gly, a la, and Ser, and the N-terminus of the amino acid sequence is that of the gene 10 leader. It was.

Plasmid pSY55B (pAR1151 of Stvdier et al., 1986) The coding sequence of the T7 RNA polymerase gene from 3128-5845) by in vitro mutagenesis of cloned DNA. I modified it. We identified the recognition sequence of restriction enzyme endonuclease NdeI as 31 It was inserted at position 71. Oligodeoxynucleotides synthesized by conventional methods was used to convert the T7 gene 1 sequence from its natural sequence TAAATG to the modified sequence CAT Changed to ATG.

Similarly, the upstream regulatory region of the Bacillus subtilis gene spoVG was transferred to the plasmid pCB1 291 [Rosenblum et al., J. Bacteriology, 148:3 41-345 (1981>), in vitro mutation at position 85 Johnson et al., Nature, 302=800- 804 (1983)), so that it also contained an NdeI cleavage site. next, The upstream regulatory sequences of the spa VG gene were isolated through these new NdeI cleavage sites. and ligated with the coding sequence of the T7 RNA polymerase gene. Escherichia coli Ha 10 After 1 transformation, restriction map the plasmid content of each ampicillin-resistant isolate. Investigated by. The correct construct was named pSY649.

spo VG: Plasmid D containing T7 RNA polymerase fusion gene NA (pSY649) is a chloramphenicol resistance gene that functions in Bacillus subtilis. Further modified to include children. First, plasmid pGR71-p43 [G Oldfarb et al., Nature, 293:309-311 (1981)] About 1200 base pairs from Nde l-5al! The fragments were separated.

This fragment contains the Bacillus subtilis p43 promoter and the adjacent sequence from Tm9. It carries the mufenicol acetyltransferase gene. Klenow DNA After filling in all sticky ends using a polymerase reaction, this fragment was pUc 13 [Veiera et al., Gene, 19:259-268 (1 It was inserted into the Xba I site in the double cloning site of 982)). Ampisi Phosphorus and chloramphenicol resistant transformants were selected for subsequent use. Ta. The correct plasmid construct was designated psY630.

Chloramphenicol acetyltrans fused to p43 fromotor sequence Sma I from plasmid psY630 containing the ferase gene = Hinc The II endonuclease cleavage fragment was gel purified and first purified using a 74 DNA polymerase. The blunt ends were ligated to the Pvu1 site of plasmid pSY649 which had been treated with enzyme. Living The modified plasmid pSY856 was transformed into Bacillus subtilis 1168. plasmid p SY856 cannot replicate autonomously in Bacillus subtilis, resulting in chloramphenicol resistance. Stable transformants of E. coli must result from integration of the plasmid into the E. coli chromosome. No [Ferrari et al., J. Bacteriology.

154:1513-1515 (1983)]. Integration phenomenon leads to homologous recombination Therefore, it is possible that 4 extension occurs at the spa VG or p43 site of the bacterial chromosome. [pSY856 is homologous to the B. subtilis chromosome only at these two sites. (contains the same NA sequence). To determine the stability of the selectable marker, the resulting rBI Strain PDL J was grown both in the presence and absence of chloramphenicol. . Expression of T4 polymerase was obtained, which affects the growth and survival of this strain. There didn't seem to be any.

Staphylococcus containing a gene encoding resistance to the antibiotic kanamycin Staphylococcusaureus plasmid p U8110 (Lancey et al., JoMed, Microbiology.

7:285-297.1974) and the growth-controlled Le5poVG of the BIPoL strain. :Used to examine the expression of T7 RNA polymerase gene. Phage T7D EcoRI-BamHI fragment of NA (2 containing the T7 gene promoter sequence) 1.402 to 22.858) was purified from plasmid pAR441 (St Udier et al., 1986). This DNA fragment was subjected to EcoR engineering and BamHI ligated into pUBllo between the side restriction enzyme endonuclease sites. positive result Mid pSY952 contains a T7-specific promoter in the same direction as the kanamycin resistance gene. Contains tar.

Plasmid pSY952 was transformed into Bacillus 168 and BIPOL. . These strains were then encoded by the plasmid-derived kanamycin virulence gene. The expression level of the coded polypeptide was analyzed. 1168, pUBllo I168 containing pSY952, BIPoL, pi! B1l From growth cultures of BIPOL containing 0 and old PoL containing pSY952, ca. 103 cells were obtained at multiple time points during the growth and sporulation cycle. these Proteins in hygrocyst samples were processed and analyzed by polyacrylamide gel electrophoresis. Analyzed.

The rate of transcription from the spo VG promoter increases as a function of cell density and The accelerated accumulation of target proteins reaches a maximum during the early sporulation period. BIPoi, containing PSY952, at a time when cultures undergo sporulation. Be expected. The results show that when a culture approaches and enters steady state, It is shown that the protein of offspring ft34kD is greatly increased. this protein The size of the kanamycin resistance gene product C3a encoded by pSY952 daie et al., J. Bacteriology, 141:117g11g2 (1980)). This protein is spoVG pSY952, which lacks the T7RNA polymerase gene controlled by 1168 or BIPOL, or lacking the T7 promoter sequence. Not found in BIPOL containing IB110. BIP containing pSY952 The maximum accumulation level of target protein after 24 hours of growth in QL is It was 20% of the cellular protein as determined by tometry.

9.b. Expression plasmid pGLO31pI of SlpI-4 in Bacillus subtilis , digested with EcoRI REN. using Klenow DNA polymerase reaction After filling in the sticky ends, the DNA was digested with BgII. plastic Sumid pSY662 was digested with Soa I and Ban+HI REN. Continued The two busulamides were separated by electrophoresis through a low melting temperature agarose gel. and purified on a NAC3 (Bi'iL) column.

The DNA fragment of pGloslpI was linked to the molecular chain of pSY662, and ampicillary E. coli was transformed in a tube (40zr/-Z). Transformants containing plasmids One (pSY662/G10/51pI-4) was analyzed for selected for the test.

Bacillus subtilis BrPoL competent cells were transformed into pSY662/G10/Sip I.

-4 and incubated at 37°C with shaking for 90 minutes. Ta. This transformation mixture was then transformed into a fresh tube containing 10 n/- tetracycline. diluted 1:100 in LB and incubated at 37°C with shaking.

A sample was taken, and the same number of cells were lysed and applied to a 5DS-PAGE separation gel. Legacy To detect the synthesis of gene 10/Slp I-4 fusion protein, anti-Slp Innoprot analysis was performed using the antibodies. 51pI-4 polypep in Bacillus subtilis Expression of cellular proteins from polyacrylamide gels to nitrocellulose After transfer to the filter, serum reactivity with anti-Slp antibody (serore act iv 1ty). Thorough fishing treatment of cells with CNBr Therefore, we show that the serum-reactive protein is the product of the 51pI-4 gene. certified. This cleaves after the methionine residue, but 51pI-4 cleaves after the methionine residue. Since it lacks, it remains in perfect condition. By CNBr treatment, More than 98% of the proteins stainable with MasiBlue dye were removed. methionine As expected for a protein lacking, 51pI-4 is intact and still Reacts with anti-3ip serum.

Example 3 Assembly and expression of S1pIIT gene 1. Overview of 51pIII gene assembly scheme Synthesis 51pIil? Zoudenshi is In the Sip I gene, and in the silk moth (Bomuyx rbOri) ), which encodes a protein similar to the crystalline region of the silk fibroin protein produced by do. Slp■ consists of the amino acid tyrosine at regular intervals (approximately 50 residues). It is more similar to silk fibroin protein. This is 51pI It is not seen in the multimer of. Sipmm story 60bp DNA's three two-pronged sex tion was chemically synthesized.

Each section was cloned by insertion into bacteriophage M13. , and the DNA sequence was confirmed. Subsequently, these sections are extracted from the vector They were taken out and concatenated together in a specific order. This approximately 180 b, p concatenation is , Slpmr monomer. The "monomers" are then linked in a specific order. , S]pIII dimers, trimers, tetramers, etc. were obtained. Next, the multimer has 5 Cloning directly into plasmid expression vectors to detect pIII protein content or first cloned into an adapter plasmid. 5IPIII Insertion of DNA into adapters enables further genetic manipulation, but this The details will be described later. The scheme of assembly is depicted as follows.

Synthesis of 2-terminal DN A section.

Section 2 5... Mountain... Song... 511.1, 121.1111. .

Section 3□5 "Monomer" Kume f2 Hit 1 2 3 = 3 DNA and S 1 p * & Mi The matching amino acid sequences of the three child sections are shown in Table 3.

The doublet DNA sequence is shown in the 5° to 3” orientation. Encoded by this sequence Amino acids (g = niblicin a = alanine, s = serine, y = tyrosine) are shown immediately below the section. Recognition sequence for restriction endonuclease cleavage are shown above each section.

The six single mRNAs mentioned above were synthesized. After synthesis, the DNA sequence is purified and homologous The strands were annealed. Approximately 1d C0,5tar) of each button, 10xA of 2I A (described) buffer and 16d sterile deionized water and 1.5-polypropylene Mixed in a manufactured Eppendorf tube. Put this tube into the beaker of the boiling water tank (II! 500-g water) for 10 minutes, then remove the beaker from the hot plate. and cooled to room temperature on the bench. This took about 1-2 hours.

Three double-stranded sections were separately cloned into M13mp18. sexy 1 of the multiple cloning sites, restriction enzymes of Smal and Bam) I Connection was made between the bare cleavage sites. Section 2 contains BamHI and PstI restriction enzymes. Connection was made between the cut sites. And section 3 contains PstI and Hindm restriction enzymes. The restriction enzyme cleavage section was used between words. Each clone is M13mp18.1, M13mp18 ．． 2, M13IIlp18.3. DNA sequence of each cloned section Decided. One representative of each section with the correct DNA sequence was collected and used for the next stage. In other words, it was used as a material in the assembly of "monomers".

3. The “monomeric” assembly NA sections 2 and 3 of 31pII were digested with restriction enzymes. The M13 clone was isolated by digestion with a That is, in section 2 digested M1301918.2 with BamHI and PstI. section In 3, M13mp18.3 was digested with PstI and HindI[I. two sections were purified and these were first digested with BamHI and HindIII. Equimolar amounts of M13mp18.1 were mixed. T. Add DNA ligase and phase Overlapping ends of the same sex were ligated in the order 1-2-3. The sticky end Due to hybridization specificity, the three sections are only effective in this order. Link.

Cloned "monomer" D in a conglomerate designated M13mp18.1.2.3 The NA sequence was found to be correct and is shown in 2 above.

4. Multimerization of “monomer” of 51pnI Preparing a large amount of “monomer” structural gene. In order to -ning. M1301P18.1.2.3 with EcoRI and old ndIn system Digested with limited enzymes. 51pII[rmonomer' was gel purified and ECORI and ligated to puc12 digested with uHindIII. Resulting plasmid D NA is prepared and the “monomer” is isolated from the vector by digestion with Ban I REN. was released and the fragment was gel purified.

To create multimers, “monomeric” DNA with Ban I ends is ligated Linked by reaction. The Ban I recognition sequence at the non-palindromic end is a head-to-tail sequence. It only allows for knots. The degree of multimerization was determined by gel electrophoresis and ethidium bromide. This was monitored by staining the DNA used.

5. Cloning of Slpm multimers Plasmid pCQV2 (Queen et al., J. AppllMol. Gen., 2:1-10 (1983)) with EcoP, I and BamHI restriction enzymes. A fragment of approximately 900 bp was purified. This DNA fragment is a batataeriophage λ cl-857 repressor gene, right promoter P8, and cro Contains genes. Plasmid pSY335 (Ferrari et al., J, Ba pJF7 in Cteriology, 161:556-562 (1985) 51] was digested with restriction enzymes EcoRI and Baa) II, and then p It was ligated to an approximately 900 bp DNA fragment of CQV2. The output obtained with this configuration is Lasmid PSY751 expresses the β-galactosidase gene at 37°C and 42°C. However, it is not expressed at 30°C (Fig. 8).

In this method, the 51pI[f gene is first inserted into an “adapter” in an intermediate plasmid. -” sequence and then subcloned into an expression system. this adapter The array has the following useful characteristics: i.e. one unique B in the center an I restriction enzyme site, 3 unique restriction enzymes on either side of Ban I elemental site, amino acids methionine, aspartic acid-proline or arginine Information that encodes proteolysis and small size. The Ban I site is Ba n is the insertion point for Slpm multimers with an I terminus.

The adapter is Applied Biosystems 380A 5ynth esizer, cloned into M13mp1g, and the DNA sequence was approved as R. Subsequently, the adapter is specifically constructed lacking a Ban I REN site. subcloned into a plasmid vector.

This receptor plasmid was prepared as follows. Plasmid pJH101 (Fe rrar i et al., 1983) was partially digested with the restriction enzyme Aha II [and Reconnected. A transformant of E. coli HB101 was transformed with chloramphenicol (11 Selection was made in a medium containing 5■/-). After restriction enzyme cleavage analysis of several isolates. :, one plasmid pSY325 was selected (Figure 7). This plasmid has , the chloramphenicol resistance gene and the origin of replication of pJHlol (pBR 322) were included. After complete digestion with Xho II, pSY325 was ligated with a gel-purified adapter. As a result, the adapter-plasmid pSY937 was obtained and this new restriction enzyme site was demonstrated.

The 51pIII multimer was cloned into the Ban1 site of pSY937 (Figure 7). ). Positive clones were identified by colony hybridization and hybridized. Use the short strand of section 1 of 51pIII as a DNA probe for diization. (probe sequences shown in Table 2). Positive clones were inserted by gel electrophoresis. It was characterized by the size of the multimers. Finally, the 51pI sequence was added to flanking adapters. Restriction enzyme sites in the target region are used to subclone into specific locations in the expression plasmid. I did a ning.

The 51pIII protein has the following amino acid composition.

slpm 1178 AA Mill 83.000 (fm) DPVVLQR RDWENPGVTQLNRLAAHPPFASDl"! GAGS (GAGA GS), GAAGY [(GAGAGS), GAAGY Co.

GAGAGSGAGAGSGAGAM Kano GRYQLSAGRYHYOLVWCQ K(fm) is a transcription start codon.

5lp1 expression vector Plasmid DNA psY1086 is a 19-repeat sequence of 51pIII <3.5 J) is a derivative of SY937 containing kb). This plasmid DNA was converted into Nru Digested with Pvu I and Pvu II, and the fragments were separated by agarose gel electrophoresis.

Subsequently, this purified 51pI[I multimer was digested with PvuI[I restriction enzyme. It was cloned into Sumid psY751. After analyzing some clones, we found that One clone (psY1008) for expression experiments and for 51pIII purification. selected.

The ampicillin drug resistance gene of psY1008 was converted into Kanama from psYlolo. Digestion of pSY633 with icine markers (Dral and SspI, and Kan” obtained by old ndIiI digestion into pUc4. Plasmid p By removing 5lpIIIe of sY1186 with Ran I, a new Plasmid psY1262 was generated. This plasmid contains A unique Ban that allows direct ligation of fragments containing the Ban I end obtained by Includes site I. This plasmid contains inserts for the following proteins: It has been used to generate plasmids. 5ELP 1.2.3 and 51p4゜ Production and purification of 51pI[I cell culture Culture E. coli in the following medium.

g/It Yeast extract 20 Casamino acid 20 Peptone 20 Gelatin peptone 20 KH,Pri42 1IP042 NaJP04+ 7H202 Cultures (500aZ~1j2/) grown overnight at 30°C were grown in 5001 fermentation volumes. 375 included in the container! It was used to inoculate the culture medium.

The fermentation vessel conditions are: tachometer reading 1100 rpm, vessel back pressure 5 psi. , ventilation rate is 170!2/min to maintain $ and melting rate over 50% 0□ It is.

OD of culture. When the value reaches 2.0, add glucose (Ig/A) and and ampicillin (0.05 g/f) were added and the OD value reached 2.0 again. 0 When D65° reached 2.0, the temperature was increased to 42°C for 10 minutes, then 3 hours for 2 hours. The temperature was lowered to 8°C. The culture was then cooled to 10°C and the cells placed in a continuous centrifuge. The cells were harvested by centrifugation and frozen at -70°C until processing.

The yields from the two separate fermentations were 7-3 kg and 5.2 kg wet weight of cells. 'Kg.

Other media are also available and impose different selection conditions on different plasmids. (i.e., kanamycin instead of ampicillin). These conditions are It was used (dose 10·1) in laboratory-scale fermentations.

It was suspended in EDTA at a concentration of 1 kg wet weight/6f. And 800 Cells were disrupted by passing through an APR cell disruptor twice at 0 ps.i. During cell lysis process , cells were kept cooled in an ice bath. The cell lysate was then transferred to a continuous centrifuge (operated at 4°C). 2 using a 5orvall RC=B refrigerated centrifuge, T2-280-tar). It was centrifuged at 6,000×G. Under these conditions, 90% of the production amount 5lf) I[I was found in the pellet. The supernatant contains the lower 8 It does not contain any products that can be recovered by NH4SO4 precipitation.

The pellets were extracted with LiBr as follows.

Method 2: Thaw frozen cells, 50 m! J Tris-HCI (p)! 7.0 ), 10 i:! f 5 mM to inhibit EDTA and protease activity ? It was suspended in MSF at a concentration of 1 kg wet weight/612. Cells buffer at room temperature for 0.5-2 hours, then lysozyme was added to a concentration of 1 g/l. and incubated for 20 minutes. Furthermore, while stirring the cell suspension, Then, β-mercaptoethanol was added to 70mM to terminate the surfactant NP40. It was added to a concentration of 1%. Subsequently, MgCb was added up to 5001M and the DNA was added at a concentration of 1/1 and incubated for 20 minutes at room temperature. moreover, The cell lysate was centrifuged according to method 1 at 26,0 OOX G using a continuous centrifuge; The upper groove was collected and passed through the continuous centrifuge once again at 26,000×G. 2 The supernatant obtained from the second centrifugation contains 5% of the total amount of terminal 51pIII. However, this is recovered with NH,SO, as described below. 1st and 2nd time 2 The pellets obtained from centrifugation at 6.0 OOX G are combined as described below. Extracted with LiBr as follows.

Method 3. In this method, the second plus encoding lysozyme of T7 phage Use an E. coli strain containing mido. This plasmid contains the 5ipnIiL gene and Compatible with plasmids encoding drug resistance determinants. This stock has the above two Grow in the same medium and under the same conditions as the method. However, T7 lysoti The cell wall is weakened by the production of 100mM EDTA and Fermentation is completed by adding NP40 at a concentration of 0.5 to 1.0% v/v. Sometimes cells can be easily lysed. Chloramphus into the fermentation medium instead of NP40 Dissolution may also be facilitated by the addition of Genicol (20ml #). a Alternatively, cells can be collected by centrifugation prior to lysis and subjected to Tri Resuspend as 1 kg wet weight/6f in s-EDTA then NP40 or It can be dissolved by adding chloramphenicol. one of them After lysing the cells using a continuous rotor method as described in the first two methods, Centrifuge the cell lysate at 26.0 OOX G.

These methods involve LiBr ejection of the pellet and NH,SO4 precipitation of the supernatant. , used for product recovery.

Purification of 5l pHl The pellets obtained by centrifugation of the cell lysate at 26,000×G as described above extract with an equal volume of 9MLiBr. Add salt solution and at room temperature Uniformly suspend the pellet by stirring. After obtaining a homogeneous suspension, this mixture Stir for 1 hour at room temperature. The mixture is then passed through a continuous rotor at 4°C or room temperature. Centrifuge at 26,000×G to obtain pellet and supernatant fractions. The upper level and resuspend the pellet in an equal volume of 9MLiBr as above. After mixing for 1 hour, The mixture was centrifuged at 26.0 OOX G, and the resulting supernatant was separated from the first LiBr extraction. The mixture was mixed with the supernatant and left overnight at 4°C. Approximately 90% of 51pI contained in cells fr is extracted with LiBr by this method.

When the LiBr extract is left at 4°C overnight, a precipitate forms, which is 26,0OOX Remove by centrifugation at G and discard. I put it in a dialysis bag and gave it some distilled water for 2 days. Dialysis is performed during intercourse.

When LiBr is removed by dialysis, the 51pIII product precipitates into the dialysis bag. . The precipitate is collected by centrifugation and washed 2-3 times with distilled water. Finally, centrifuge the washed product Freeze dry.

To recover Slpm from the 26.000 x G supernatant fraction, use NH, S04 precipitation. The lees are used. This reaches 38% saturation of solid NH, SO, <231 g/Il) to the sample kept at 4°C. Subsequently, the mixture was heated to 4°C. Stir for 2 to 3 hours. The precipitate was collected by centrifugation using a continuous centrifuge, and an equal amount was distilled. Wash with water or 0.5% SOS aqueous solution. This pellet is removed by continuous washing. It increases its whiteness and is removed as a contaminant protein. S]pIII is a washed pellet and can be recovered and dried by freeze-drying.

Slpm trypsinization process 51pIII, 50 mM Tris-HCI (pH 8,0), IM N aC] Suspended in buffer solution and placed in a 37°C water bath, TPCK)! J psin treatment solution mixed into a suspension. Final trypsin concentration was 0.1%.

After 3 hours, the solution was centrifuged at 16.0 OOX G for 15 minutes, and the pellet was initially % SDS solution followed by distilled water. After each wash, pellet The samples were collected by centrifugation. The final product was suspended in water and further dialysis was continued at 4°C.

By trypsin treatment, 51pIIr was purified to a purity of 99.4%. Physical measurements of protein are based on naturally occurring repeating amino acid polymers produced microbially. The antiparallel antiparallel beta sheets of the crystalline regions of silk fibroin accurately characterize the existing polymers. Experimental models of structure [Marsh, Corey and Pauling, Bi ochem.

Biophys, Acta, (1955) 16; Pauling, and and Corey, Proc.

Natl, Acad, Sci, USA (1953) 39:247). It was done to recognize the Preliminary analysis of the X-ray analysis image obtained from Sip mill film is as follows: As described by Fraser, MacRai, and Steward (1966). It matches what is listed (Table 4). Circular dichroism (CD) and polarization of Slpm FTIR spectral analysis results show beta and inverse beta structures. coincides with the altitude spread of .

In various solvents, the spectra obtained from 51plII and the naturally occurring Compare with that of silk fibroin (Isuka and Young, Proc, N atl, 'Acad, Sci, USA (1966) 55:1175) , 51 pI in solution [[ is the random structure and regular structure found in silk fibroin. It has been shown that it consists of a mixture of highly specific structures (AG)ゎゎ　9.42　6.95　 8.87 (AGAGSG)ゎ 9.39 6.85 9.05 CTP fraction 9. 38 6.87 9.13 Natural fibroin 9.40 6.97 9.209 , 44 6.95 9.30 31 pI [I 9,38 6.94 8.97 Six types of oligonucleotides were prepared as described above. Kreokudojin was synthesized and purified.

Add oligonucleotides a(iii), (iv), (V) and (vi) Plasmid pBsm1 kneeled and digested with old ndIII and EcoRI 3 (+) was connected to DN, A (Stratagene). of ligation reaction The product was transformed into E. coli strain JM 109. A colony of transformants was Selected by ampicillin resistance. 32P-labeled oligonucleotide (ii) , (iv) Screen colonies by hybridization with did. Plasmid DNA from some hybridization positive clones was purified and its nucleotide sequence was determined. Two of these plasmids, pSY1 292 and pSY 1293, oligonucleotide (in), (iv), The sequences shown in (V) and (vi) were included. These arrays contain one Contains all nucleotides present in synthetic oligonucleotides except Ta. There was one in which the 7th G20 base pair was missing (iii).

Due to this base pair deletion, one Ban T site became non-functional.

To introduce a second BanU at the 5′ end of the gene fragment, an oligonucleotide was used. Annealing of codes (i) and (ii) and erasing with old ndIII and EcoRI. It was ligated to the converted plasmid pBsm13(+). Transformation for ampicillin resistance Plasmid DNA from the body was purified. Two plasmids, pSY1295j, - and pSY 1296 C5tul (units included in the oligonucleotide sequence) The sequence of the protein that can be digested at the unique site (neak site) was determined.

Both plasmids contain sequences represented by oligonucleotides (i)g and (ii). It was shown that Plasmid DNA from pSY1292 was converted into the former ndIII , SI nuclease, and ECORI. Agarose the digestion products Separate by gel electrophoresis and cut out a DNA fragment of approximately 150 base pairs from the gel. did.

Plasmid DNA obtained by digesting this DNA fragment with StuI and EcoRI It was ligated with pSY1296. This ligation reaction product was added to E. coli JM strain 109. Transformed and selected for ampicillin resistance.

A column that hybridizes with the 32p-labeled oligonucleotide (V) I chose knee. Plasmids from two hybridization positive clones The DNA was purified and sequenced. These plasmids were transformed into pSY12 97 and pSY129g. These contained the following sequences .

Ko Modified to accept A fragment. Two oligonucleotides and Bam1 I The plasmid DNA psY937 was ligated to the digested plasmid DNA psY937.

The product of the ligation reaction was transformed into E. coli strain JM 109. Ampicillin resistance Colonies of transformants showing the following were selected. 32p labeled oligonucleotide ( Colonies were screened by hybridization with vii) . According to the determined DNA base sequence, there are two hybridization positive clones. pSY1299 and pSY1300 contain oligonucleotides (v ii) and (vnI) were included.

Digest plasmid DNA, pSY1298, with Ban II, and assemble the digested fragment. Separated by galose gel electrophoresis. Excise an approximately 150bp EBSI gene fragment and purified by electrophoresis and ethanol precipitation. The size is 450bp. Purified fragments of approximately IK were self-ligated to induce multimers in the 6.000bp range. did. The self-ligated product was then transformed into a Ban II digested plasmid [1 It was ligated to NApSY1299. This ligation reaction product was transferred to E. coli HB101 transformed into a strain. Transformants resistant to chloramphenicol were selected. Each trait Plasmid DNA from the transformant was purified and cyclized by εBSI multimer DNA insertion. We analyzed the increase in Size ranges from 1.5 kbp to 4.4 kb 10 clones with inserts spanning p (psY1240-psY124 9) was obtained.

Expression of EBSI multimer gene One of these clones, pSY1248, contained a 4kb EBSI multimer. This gene was recloned into the λPR expression vector psY751. Ta. Plasmid DNA from pSY1248 was transformed into NruI and pvuI Digested with f and separated by agarose gel electrophoresis, the EBSI multimer gene The DNA band was excised, sampled and purified by NACS purification. plasmid psY? DN A from 51 was digested with Pvu II and the Nuv I-Pvu [ligated with fragment. The ligation reaction product was transformed into E. coli HB101. , and ampicillin-resistant transformants were selected. New plasmid pSY Two clones containing 1280 were selected. E. coli cells containing pSY1280 at 30°C and OD6°. Grow to a value of 0.7 and then heat to 42°C for 1.5 hours. The temperature was raised. Proteins produced by cells are analyzed by 5OS-PAGE. analyzed. The separated proteins were transferred to nitrocellulose paper and Detection was by immunoreactivity using LP rabbit serum. Apparent molecular weight 120k A strongly reactive protein band with D was observed.

The ampicillin drug resistance gene of psy 1280 was replaced with kanamycin marker. The resulting plasmid was called pSY1322. This plasmid is EBSI Used in fermentation for purification (see Methods).

psY1332/psY1280 EBSI protein 1413 AA Ml li 113.159MDPVVLORRDWBNPGVTQLNRLAAHP PFASERFCMGS [(GVGVP), (GAGAGSGAGAGS), ] 2! MCYRA) IGYQLSAGRYHYQLVWCQKEBSI Tampa Purification of wood grains Fermenting E. coli HB 101 strain containing plasmid Psy 1280 at a volume of 101 I let it happen. The cells were concentrated by filtration and harvested by centrifugation. Peret The transformed cells were stored frozen at -70°C until processing. Thaw frozen cells on ice and 50 mM Tris-HCI (pH 7,0) per g wet weight of cells. , 10mM EDTA.

5 (IIMPMSF).The cells were suspended in a solution containing IIMPMSF. The mixture was crushed twice at 15.0 OOpsi and cooled to 0°C. crude lysed product Cleared by centrifugation at 26'X G for 20 minutes. This supernatant protein is By adding solid ammonia sulfate to 20% saturation (114 g/f) It precipitated.

The precipitate was collected by centrifugation in IQK XG for 10 minutes. 10 berets - resuspended in 10mM Tris (pH 8,0), 15M Na at 4°C. Dialyzed against C1. The dialysis solution was treated with 0.1% trypsin (S) for 1.5 h at room temperature. igma) and reprecipitated using 20% ammonium sulfate. Shen The precipitated protein was resuspended in water and incubated at 4°C for 10 d in Tris(p) 17.0). , and dialyzed against 1mM EDTA solution. The protein purity of this sample is It was determined to be 83% by analysis of the amino acid, iiffl.

Elastic properties of EBSI proteins A soluble preparation of the semi-purified EBSI protein described above was incubated at 37°C for 30 min. The mixture was incubated and centrifuged at 10K x G for 10 minutes at room temperature. This process The EBSI protein aggregates and becomes insolubilized, and the pellet becomes a translucent solid. Become. This solid resisted mechanical fragmentation by swirling or softening with a glass rod. child The solid could be cut with a razor into strips exhibiting high elasticity. child They perfectly retained their shape after repeated stretching and relaxation. and compress It also withstood a lot of stress and did not cause any irreversible deformation of the structure.

Purification of EBSI EBSI samples (-70% purity) were treated with 50mM Tris-HCI for 15 minutes at 4°C. , 50mM NaCl, (pH 8,0) buffer solution overnight. The liquid was replaced once. If a precipitate is observed, place the sample at 4°C for 15 minutes at 27°C. ．． Centrifugation was performed at 000X G. The supernatant was added to 50mM Tris-HC. I, 50mM NaCl, DEA equilibrated with (pH 8,0) buffer It was applied to R-5ephace 1 column. Collect the effluent from the fractions containing EBSI. I played pool for a while.

The pooled fractions from the DEAE-Sephacel column had a final concentration of 2 in the sample. NaCl was added to give M. Insoluble substances at 27,000x for 20 minutes It was removed by centrifugation at G. This supernatant was then diluted with 5 ml of 2M NaCl. Phenyl-Sepha equilibrated with 0mM phosphate pH7 buffer applied to a rose column. Eluted protein is no longer detected with A21o The column was thoroughly washed with buffer until Next, 50o+M! Sodium chloride Elute from the column by applying a stepwise concentration gradient with 100% sodium chloride (pH 7.0) buffer. It was. East active fractions were pooled and stored at 4°C for subsequent analysis.

By adding these steps to the above method, 100% pure EBSI can be obtained. I was caught.

Two oligonucleotide chains were synthesized as described in the Methods section. , and purified.

The two oligonucleotide buttons were annealed to form REN's Hindm and Ligate using the DNA of plasmid pBsm13 (+) digested with EcoRI. I set it.

This ligation reaction product was transformed into E. coli JM 109 strain. Transformant colo Hybridization with 32p-labeled oligonucleotide (i) Screened by. Positive from hybridization positive colonies Mid DNA was purified and sequenced. One plasmid, pSY1287 contains the sequences shown for oligonucleotides (i) and (ii) there was.

Plasmid DNA from pSY1287 was digested with Ban I restriction enzyme. Fragments were separated by agarose gel electrophoresis.

An approximately 60 bp ELPI gene fragment was excised and purified using a NACS column. Ta. Purified fragments of approximately IK were converted into multimers ranging in size from 300 bp to 5000 bp. self-ligated to produce .

Subsequently, the self-ligation product was digested with plasmid DNA using Ban I restriction enzyme. It was ligated using psY937. The product of this ligation reaction was transformed into E. coli HB101 strain. transformed into. Transformants resistant to chloramphenicol were selected. individual shapes The plasmid DNA of the transformant was purified and the size determined by the ELPI overlapping DNA insert. We analyzed the increase in The size is 1. Okbp to 2.5 kbp Four clones (pSY138g-1391) within the range were obtained.

These clones were recloned into the λPR expression vector PSY751. child The clone thus obtained (pSY1392-1395) was used to express ELPI. used.

The amino acid composition of ELPI protein was as follows.

psY1395 ELPI protein 859 AA MW72,555MDP VVLQRRDIIiENPGVTOLNRLAAHPPFARN I LA IRW [(VPGVG)4,,VPWTRVDLSAGRY)! YQL VIICOKSELP 1 gene composition and expression As described in the “Methods” section, two types of oligonucleotides were used. Plasmid pSY annealed in the otide region and digested with Pst I restriction enzyme 1304 (pSY1304 is a substitute for the trimer carrier of pSY857) It differs from pSY857 in that it has a monomer unit in Chloramphenico Plasmid DNA was purified from the cell-resistant transformant colonies. restriction enzyme 5n When we sequenced psy1365, a plasmid digestible with aB I, we found that the This was proven to be true.

The purified ELPI gene was produced as described above (t, LPI construction and expression). Mung Bea as described by Stratagene n) was treated with nuclease. Subsequently, the mixture of DNA fragments was treated with FspI , 5naBI, # and bovine intestinal phosphatase. This series The product of the ligation reaction was transformed into E. coli HB 101 strain, and chloramphenicol A resistant strain was selected. Plasmid DNA from individual transformants was purified and EL An analysis of the DNA insertion site of the P'I monomer was performed. Two plasmids, psY The sequences of 1366A and psY1366B were determined. Both are ELPI D It was shown to contain the NA sequence in the correct orientation.

Plasmid DNA 5pSY1366 was digested with Ban I restriction enzyme and 5EL The DNA fragment containing F1 was gel purified. To create multimers, 5E of 1; The DNA fragments of LP1 were self-ligated. Size is 500bp to 10kbp Multimers in the range of .

5ELP1 multimer was cloned into the BanIR position of pSY1262. .

Positive clones were characterized by electrophoresis for inserted multimers and expressed. and used for protein analysis.

PSY1396 5ELPI protein 2025 AA MII1148,2 12MDPVVLORRDWENPGVTQLNRLAAHPPFASDPMG AGS (GAGAGS) g [GA’A (VPGVG) 4VAAGY (GAGA GS)s] 24GAA (VPGVG), VAAGY (GAGAGS) 2GAGAM[1PGRYQLSAGRYHYQLVlIC(IKSELP) 2- Monomer composition Digest 129 g of plasmid DNA 5pSY with Bann restriction enzyme and transfer to EBS. The I gene fragment was purified as described above. The EBSI monomer fragment was pSY13 digested with restriction enzymes and treated with bovine intestinal phosphatase 04 (pSY9 containing monomer of 51pIII, constructed as pSY857) 3? ).

The mixture of ligation products was transformed into E. coli strain HB101. Chloramphu Transformants resistant to genicol were selected. After restriction enzyme analysis of several isolates , one plasmid pSY containing a DNA fragment corresponding to the EBSI monomer gene 1301 was selected.

5ELP 2-Duplicate gene assembly and expression plasmid DNA, pSY 130 1 was digested with Ban I restriction enzyme to obtain a DNA fragment containing "5ELP2 r monomer". Gel purified. To create multimers, 1 g of 5ELP2 DNA fragment was Connected.

Multimers with sizes exceeding 12 kbp were obtained.

The 5ELF2 multimer was cloned into the Ban I site of pSY1262.

Positive clones were characterized by gel electrophoresis for the size of the inserted multimer. I got it. Clocks with inserts ranging in size from 1.5 kb to 1 lkb selected. Plasmid DNA 5pSY containing a 6kb (18 repeats) insert 1372 was used for further analysis and protein purification.

5ELP 2 protein purification Escherichia coli HB 101 strain containing plasmid psy 1372 was described using the “fermentation method”. It was fermented according to the method. The cells were collected by centrifugation. pelletized fine Cells were stored frozen at -70°C until processing. Thaw the frozen cells on ice and 50mM Tris-HCI (pH 7,0), 10mM per g wet weight It was suspended in a solution containing EDTA and 5dPMSP. The cell is (, aul The cells were disrupted by passing through an in-cell disruption device at 8,000 psi. Crude cell lysate production The material was clarified by centrifugation at 26,000×G for 20 minutes. This supernatant is Contains over 75% of 5ELP 2 and 20% ammonia sulfate (114g) /f') was precipitated. The precipitate was heated at 10.0 OOX G for 10 minutes. was collected by centrifugation. Resuspend the pellet in 10-mM water at 4°C. Dialysis was performed against Tris (pH 8,0), 0.15M NaCl. Approximately 10% To recover the insoluble protein fraction containing the SEI, P2 protein, The analyzed material was centrifuged at 26.0 OOx G for 15 minutes. This insoluble protein The pellets were washed twice in 0.2% SDS for 30 minutes at 50°C with occasional stirring. Purified. The insoluble protein was centrifuged for 15 minutes at 26,000×G. It was collected every time and washed with 50% ethanol. Re-hydrate the final protein pellet in water. It was suspended and analyzed by Western blot analysis and amino acid composition. Vestal According to the plot, the 5ELP2 protein has a large molecular weight (>150k D) appears to have a uniform size consistent with . In terms of amino acid composition, 5ELF 2 The preparation has approximately 80% purity and the observed molar ratio of amino acids (Ser:Gly:Ala:Pro:Val:Tyr) is in pSY 1372 The composition value is very close to the expected composition value expected from the existing SεLP2 sequence.

psY1372 5ELP2 protein 2055 AA sin 152.354M DPVVLORRDWE)IPGVDINGOLNRLAA)IPPFASDPMGA GS (GAGAGS) 2 (GVGVP).

[(GAGAGS) -GAAGY (GAGAGS)s (GVGVP) -] 1t (GAGAGS), GAAGY (GAGAGS) 2GAGAMD PGRYOLSAGRYHYQLVWCQKSELP 3-Constitution and expression Plasmid DNA pSY1301 was partially digested with Hae II restriction enzyme, Digested fragments were separated by agarose gel electrophoresis. the larger DNA fragment It was excised and purified with a NACS column. The purified fragments are self-ligated and the ligation reaction is T4DNA IJ gauze was heated at 70°C for 15 seconds to inactivate it, and finally P Digested with stI restriction enzyme. Subsequently, the digestion mixture was transformed into E. coli i 109 strain. It was transformed. Transformants resistant to chloramphenicol were selected. individual transformation Plasmid DNA from the molt was purified and the following analysis was performed (1) Pst I restriction enzyme resistance, and (2) 5ELP2 gene dislocation locus 60 Deletion of the bp Hae II fragment. One clone (pSY137?) has both met the requirements. Plasmid DNA psy 1377, Ban I system The DNA fragment was digested with a limiting enzyme and gel-purified containing 5ELP3 monomer. multimer The 5ELP3 DNA fragments of 1z were self-ligated to create 1z.

Multimers ranging in size from 500b11 to 10 kbp were obtained. 5EL The P3 multimer was cloned into the Ban I site of pSY1262. positive The loans were characterized by electrophoresis for the size of the inserted multimer, and Used for expression and protein analysis.

psY1397 5ELP3 Protein 2257 AA MW 168 ．． 535MDPVVLQRRDWENPGVTQLNRLAAHPPFAASDP MGAGS (GAGAGS).

[(GVGVP)-(GAGAGS)-] 24(GVGVP), (GAGAG S),GAGAMDPGRYQLSAGRYHYQLVWCQKSLP4-Configuration and expression Plasmid DNA pSY1304 (different from the trimer unit of pSY857) psY857), which has one monomer unit, was partially depleted with Hae II. and the digested fragments were separated by agarose gel electrophoresis. the larger one The DNA fragment was excised and purified with a NACS column. The purified fragments are self-ligated, The ligation reaction was then heated at 70°C for 15 seconds to inactivate the 74 DNA ligase. The mixture was heated and finally digested with PstI restriction enzyme. Subsequently, the digestion mixture was transformed into E. coli JM 109 strain was transformed. Select chloramphenicol-resistant transformants Ta. Plasmid DNA from each transformant was purified and subjected to the following analysis.

(1) Resistance to Pst I restriction enzyme, and (2) 5ELP2 gene Deletion of a 60 bp HaelI fragment at the transgenic locus. One clone (pSY 137g) met both requirements. Plasmid DNA pSY 13 78 was digested with Ban I restriction enzyme to obtain a DNA fragment containing 5LP4 monomer. was gel purified. To create multimers, the 5LP4 DNA fragments of 1z were self-linked. I tied it. Multimers ranging in size from 300 bp to 6 kbp were obtained. . The 5LP4 multimer was cloned into the Ban1 site of PSY1262. Yang characterizing the sexual clones by electrophoresis for the size of the inserted multimer; and for expression and protein analysis.

psY1398 5LP4 protein 1101 AA Mill 76.23 1MDPVVLORRDWENPGVTQLNRLAAHPPFASDPMGA GS [(GAGAGS) G co, 7 (GAGAGS) 4GAGAMDPGR YQLSAGRYHYQLVWCQK As the above results show, highly repetitive sequences can be prepared and cloned, including silk and other proteins, and antigens. This iteration for expression can produce a variety of products that can mimic natural proteins. An array is used. Furthermore, the peptides under inducible conditions in various hosts. A novel system for controlling expression is provided. Thus, presenting novel protein-like products. can be used to demonstrate new properties or to improve the properties of naturally occurring products. The possibility of sexual proximity arises.

Literature 1, Maniatis, ↑+I Fr1tsch, E, F, and Sambro ok, J, 1982゜Mo1ecular Cloning: A Lab oratory Manual, Col1d Springl (arbor La laboratory, Colorado Spring Harbor, NY.

2, Laemmli, U., 1970. Nature (London) 227:680-685゜3, Applied Biosystems Ll ser Bulletin, 1984. No, 13°4, Matteucci , M.D., and Garuthers, M.H., 1981゜Journal. Amer, Che+n, 5oc0. 103:3185-3319゜5, McB ride, L.J., and Caruthers, M.L. (, 1933°Tet rahedron Letters, 24:245-248°6, Sm1th, 19800Methods in Enzymology, 65:371-37 9゜Bacteriol, 81741-746゜9, Davanloo, P., Rosenberg, A., HoDunn, J., and 5tud. ier.

F.W., 1984. Proc, Natl, Acad, Sci, USA, 81:2035-2039゜10, Rosenbluh, A., Bann er, C.D.B., Losick, R., and Fitz-James, P. , C, 1981, J, Bacteriol, 148:341-351°12, Queen, C01983, J, Applied Mo1ecular Gen etics, 2:14, Johnson, %II, C0, Moran, C, P, and Losick, T, R01983゜Nature (London), 302:800-804°15, 5tudier, W, F, and Moffat , B.A., 1986. J, Mol, Biol.

189:113-130° 16, Goldfarb, D., S., Doi, R.H., and Rodr. iguez, R, L, 1981° Nature (London), 293: 309-311゜17, Ferrari, F, A, Nguyen, A , Lang, D., & Hoch, J.A.

1983, J. Bacteriol, 154:1513-1515゜18. Lacey, R.W., and Chopra, 1.1974. J, Medo Microbiology.

? :285-297° 19. Norrander, J., Kempe, T., and Messin. g, J, 1983゜Gene, 26:101-106゜ 20. Sanger, F., N1cklen, S., and Coulson , A, R, 1977° Proc, Natl, Acad, Sci, USA, 74 :5463-5467゜21,81gg1n, M, D, , Gibson, T, J., and Hong, G. P., 1983-Proc, Natl, Acac. ! , Sci, USA, 80:3963-3965°22, Zagursky, R, J., Baumeister, K., Lomax, N., and Be. rman, M.L., 1985. Gene Anal, Tect+r+, 2: 89-94゜23, Sanger, F., and Coulson, A.R., 1 978, FEBS Letters.

87:107-110° 24.5adler, J., Ro, Techlenburg, M., and J. L, Betz, 1980゜Plasmids containing m any tandem copies of a 5yntheticlact ose operator, Gene 8:279-300 = described herein All publications and patent applications published herein are based on the expertise of persons skilled in the art to which the present invention pertains. It indicates the level. All publications and patent applications are To the extent that a patent application is specifically and separately designated for bibliographical incorporation, It has been incorporated as a document.

Once the invention has been fully described, it will be helpful to those skilled in the art to refer to the attached Various changes and modifications may be made thereto without departing from the spirit and scope of the claims. It is obvious that it can be done.

Engraving (contents (no changes)) FIG.1 FIG. 2 A Immunotubule 8kD using antibody against β-lactamase 1 1 2 3 4 5 6 7 FIG.3 FIG.4 A T7gp 10/5lLk 1 fusion: Immubutu0tuto using anti-silk Ab -----・1#ll11-m--18kD conductor conductor mosquito l8 FIG.6 Blpol　Blpol/ρG9c3 MW 1 2 3 4 5 6 MW1 2 3 4 5 6 FIG, 8 FIG.9 Aβ-galactosidase/Stp III fusion: Amidopura Tsuku staining procedure supplement Orthographic (method) April 4, 1999 Yoshi, Commissioner of the Patent Office 1) Takeshi Moon 1.Display of the incident PCT/US87102822 2. Name of the invention Construction of synthetic NA and its use in the synthesis of large polypeptides 3. Person who makes corrections Relationship to the incident: Patent applicant Name Jintro Corporation 4. Agent Address: 8-10-5 Toranomon-chome, Minato-ku, Tokyo 105, Date of amendment order March 7, 1999 (shipment date) 6. Subject of correction (1) “Representative of patent applicant” in writing pursuant to Article 184-5, Paragraph 1 of the Patent Law column (2) Power of attorney (3) Translation of the specification (4) Translation of the scope of claims (5) Translation of drawings 7. Contents of correction (1) (2) As per attached sheet (3) Translation of the specification (no change in content) (4) Translation of the scope of claims (No change in content) (5) Engraving of the translation of the drawing (No change in content) 8, Attachments catalog of types (1) Amended Article 184-5 of the Patent Act One document pursuant to the provisions of paragraph 1 (2) Power of attorney and its translation: one copy each (3) One translation of the specification (4) One translation of the scope of claims (5) One engraving of the translation of the drawing lan dyxi trial review I proboscis crow 1411・-＾11 police proboscis crow-III11・PCτ/Use fu 1028 22

Claims (1)

Claims: (1) A DNA sequence encoding a peptide comprising a repeating unit of an oligopeptide, the repeating unit comprising at least three different amino acids and a total of four The beftide is characterized by containing 30 amino acids, and at least two amino acids are present in the beftide. repeating units of repeating units and at least two identical amino acids in each repeating unit, said units may be connected by amino acid bridges of about 1 to 15 amino acids, said DNA sequence comprising: The following formula: Kk(WMXxNYy)iLl {where K and L are each a DNA sequence encoding an amino acid sequence consisting of about 1 to 100 amino acids, and K and L are less than about 20% of the total amino acids. , k and 4 represent 0 or 1, and W has the following formula: [(A)n(B)P]q, where A is a DNA sequence encoding the oligopeptide repeating unit. comprising about 12 to 90 nucleotides in said repeating unit, wherein at least two codons encoding said same amino acid present in said repeating unit are different, wherein there is a difference in at least one nucleotide. There are at least two different A's, and B is the same or different unit and has about 3 to 45 nucleotides and is distinct from A, encoding an oligopeptide unit other than that encoded by the A unit. n is an integer ranging from 1 to 100, and each p is 0 or 1, respectively. and q is at least 1 and selected to form a DNA sequence of at least 90 nucleotides), and M and N are the same or different. A codon DNA sequence consisting of 0 to 18 nucleotides in reading frame with W and X, respectively, where X may be the same as W or different. , has the following formula: [(Al)n1(B1)p l]q1, Y may be the same as or different from W, and has the following formula: [(A2)n2(B2)P2]q2 (wherein all symbols follow the definitions of their corresponding letters), x and y are 0 or 1, i is 1 to 100, and the sum of q・q1 and q2 is at least 1. and approximately 50 or less} A sequence of DNAs characterized by the following. (2) The DNA sequence according to claim 1, wherein the DNA sequence has about 10 kilonucleotides or less. (3) The DNA sequence according to claim 2, wherein M and N have 0 nucleotides, and at least one of x and y is 0. (4) A DNA sequence encoding a peptide comprising a repeating unit of an oligopeptide, the repeating unit comprising at least three different amino acids and a total of four The peptide is characterized by containing 30 amino acids, and at least two amino acids are present in the peptide. repeating units of repeating units and at least two identical amino acids in each repeating unit, said units may be connected by amino acid bridges of about 1 to 15 amino acids, said DNA sequence comprising: The following formula: Kk[(A)n(B)P]qLl {where K and L are each a DNA sequence encoding an amino acid sequence consisting of about 1 to 100 amino acids, and K and L are less than about 20% of the total amino acids, k and l represent 0 or 1, and A is a DNA sequence encoding said oligopeptide repeating unit, comprising about 12 to 90 nucleotides in said repeating unit. at least two codons encoding said same amino acids that are present in said repeats are different, in which there are at least two different A's that differ by at least one nucleotide, and B is the same or different unit and has about 3 to 45 nucleotides, and the oligopeptide is encoded by the A unit. Codes other than units, is different from A. n is an integer ranging from 1 to 100, and p is 0 or 1, respectively. and q is at least 1 and selected to form a DNA sequence of at least 90 nucleotides. (5) said A is the same amino acid at least three times in at least two different codons; 5. A DNA sequence according to claim 4, which encodes a noic acid. (6) The DNA sequence according to claim 4, wherein the same amino acid is glycine. (7) A DNA sequence encoding a peptide containing a repeating unit of an oligopeptide, characterized in that the repeating unit contains the amino acid sequence GAGAGS or GVGVP. and the DNA sequence has the following formula: [(A)n(B)P]q {wherein A is a DNA sequence encoding the oligopeptide repeating unit, which is the same or differ in at least two G codons among the different A, where there are fewer There are at least two different A's, both differing by one nucleotide, and B's are the same or different units, having about 3 to 45 nucleotides, other than the oligopeptide unit encoded by the A unit. code, which is different from A a DNA sequence of at least 90 nucleotides, n is an integer ranging from 5 to 25, each p is 0 or 1, and q is at least 1; a DNA sequence selected to form }. {8) The DNA sequence of claim 7, wherein the oligopeptide is GAGAGS and B comprises a tyrosine codon. (9) The DNA sequence according to claim 7, wherein A has the following formula: GGGCGGGCGCAGGAAGT. (10) The DNA sequence according to claim 8, wherein A comprises the sequence: [there is a sequence]. (11) The DNA sequence according to claim 7, wherein the oligopeptide is GVGVP. (12) The DNA sequence according to claim 11, wherein A has the following formula: GGTGTAGGCGTTCCG. (13) The DNA sequence according to claim 1, wherein WMX comprises the sequence: [there is a sequence]. (14) A polypeptide comprising a recombinant expression product of a DNA sequence encoding a peptide having repeating units of an oligopeptide, the oligopeptide comprising at least three different amino acid molecules and a total of four or 30 molecules of a characterized in that it contains a amino acid, and there are at least two repeating units in the peptide. and there are at least two identical amino acids in each repeating unit, the units being joined by an amino acid bridge of about 1 to 15 amino acids, and the DNA sequence has the following formula: Kk (WMXxNYy)iLl {where K and L are each a DNA sequence encoding an amino acid sequence consisting of about 1 to 100 amino acids, K and L are less than about 20% of the total amino acids, and k and 1 represents O or 1, W represents the following formula: [(A)n(B)P]q (wherein A is a DNA sequence encoding the oligopeptide repeating unit, and the repeating unit at least two different codons encoding the same amino acid present in the repeat unit, wherein at least two different codons differ in at least one nucleotide; A is present, and B is the same or different unit, having about 3 to 45 nucleotides and encoding an oligopeptide unit other than that encoded by the A unit, which is distinct from A. n is an integer ranging from 1 to 100, and each p is 0 or 1, respectively. and q is at least 1 and selected to form a DNA sequence of at least 90 nucleotides). Chido. (15) The polypeptide according to claim 14, wherein the polypeptide contains at least one of GAGAGS and GVGVP as a repeating unit. (16) A peptide comprising a recombinant expression product of a DNA sequence encoding a peptide having a repeating unit of an oligopeptide, the peptide comprising a small amount of the oligopeptide. characterized in that it contains at least three different amino acids and a total of 4 to 30 amino acids, and that at least two repeating units are present in said peptide; at least two identical amino acids are present in each repeating unit; and may be linked by an amino acid bridge made of 15 amino acids, such that the DNA sequence has the following formula: A DNA sequence encoding an amino acid sequence consisting of amino acids, wherein K and L represent less than about 20% of the total amino acids, k and l represent 0 or 1, and W represents the following formula: [(A)n (B)P]q (wherein A is a DNA sequence encoding the oligopeptide repeating unit, comprising about 12 to 90 nucleotides in the repeating unit, at least two different codons encoding the same amino acid, in which there are at least two different A's differing in at least one nucleotide, and B's are the same or different units, about 3 to 45 nucleotides and encodes an oligopeptide unit other than the oligopeptide unit encoded by the A unit, which is different from A. n is an integer ranging from 1 to 100, and each p is 0 or 1, respectively. and q is at least 1 and selected to form a DNA sequence of at least 90 nucleotides). (17) The port according to claim 16, wherein A codes GAGAGS or GVGVP. Ripeptide. (18) A control system for regulated transcription in a prokaryotic host, comprising: [1] a structural gene encoding an RNA polymerase foreign to said host capable of transcribing DNA into messenger RNA; consists of an inducible promoter [2] A polypeptide of interest under the transcriptional control of a promoter that does not function with the endogenous RNA polymerase of the host but functions with the exogenous RNA polymerase. A control system comprising a structural gene encoding a code. (19) The inducible promoter is a β-galactosidase promoter or The control system according to claim 18, which is a spoVG promoter or a spoVG promoter. (20) A prokaryotic host comprising the DNA sequence according to claim 1. (21) A method for producing a peptide comprising a recombinant DNA expression product of a DNA sequence encoding a peptide comprising a repeating unit of an oligopeptide, the method comprising: characterized in that each position contains at least 3 different amino acids and a total of 4 to 30 amino acid molecules, there are at least two repeating units in said peptide and at least two identical amino acids in each repeating unit. exists and the units are connected by an amino acid bridge made of about 1 to 15 amino acids. and the DNA sequence has the following formula: Kk(WMXxNYy)iLl {where K and L are each a DNA sequence encoding an amino acid sequence consisting of about 1 to 100 amino acids; and L are less than about 20% of the total amino acids, k and l represent 0 or 1, and W is of the formula: [(A)n(B)P]q, where A is a DNA sequence encoding a peptide repeat unit, comprising about 12 to 90 nucleotides in the repeat unit, wherein at least two codons encoding the same amino acid present in the repeat unit differ; there are at least two different A's that differ in at least one nucleotide, and B is the same or different unit and has about 3 to 45 nucleotides, and the oligopeptide encoded by the A unit. Codes other than units, is different from A. n is an integer ranging from 1 to 100, and each p is 0 or 1, respectively. and q is at least 1 and selected to form a DNA sequence of at least 90 nucleotides), M and N may be the same or different, and q is at least 1 and is selected to form a DNA sequence of at least 90 nucleotides; A DNA sequence consisting of 0 to 18 nucleotide residues in reading frame with W and X, respectively, where X may be the same as or different from W, and has the following formula: [ (A1)n1(B1)p1]q1, Y may be the same as or different from W, and has the following formula: [(A2)n2(B2)P2]q2 (wherein all symbols (according to the definitions of their corresponding letters), x and y are 0 or 1, i is 1 to 100, and the sum of q.q1 and q2 is at least 1 and not more than about 50. wherein the method comprises growing a prokaryotic host according to claim 20, wherein the DNA sequence is under the transcriptional and translational control of a functional transcription initiation and termination control region in the host. , thereby expressing the peptide and isolating the expression product. A method comprising and. (22) The method according to claim 21, wherein the peptide comprises at least one of repeating units GAGAGS and GVGVP.